Manipulation of sucrose synthase genes to improve stalk and grain quality

ABSTRACT

The invention provides a novel isolated sucrose synthase nucleic acid and its encoded protein. The present invention also provides methods and compositions relating to altering sucrose synthase levels in plants, and in particular, in plant stalks and/or plant seeds. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and incorporates byreference U.S. Provisional Application No. 60/270,777, filed Feb. 22,2001.

FIELD OF THE INVENTION

[0002] The present invention relates generally to plant molecularbiology. More specifically, it relates to nucleic acids and methods formodulating their expression in plants.

BACKGROUND OF THE INVENTION

[0003] Chemical composition and mechanical properties of plant materialsdetermine to a major extent how those plant materials are utilized. Cellwall content and composition account for most of the variation inmechanical strength of plant tissues. Also, cell wall composition is amajor determinant of silage quality. Cell walls constitute a major sinkin the vegetative parts of plants, accounting, for example, forapproximately 80% of the corn stalk (FIG. 1). For the whole corn plant,including grain, cell wall accounts for approximately 35-40% of the drymass.

[0004] Cellulose, the most abundant organic molecule on Earth, is madeat the plasma membrane and directly deposited into the cell wall [Ray,et al., (1976), Ber. Deutsch. Bot. Ges. Bd. 89:121-146]. By inter- andintra-chain hydrogen bonding, β-1,4-glucan chains form para-crystallinemicrofibrils which eventually form ribbons and fibers, giving cellulosea very high tensile strength [Niklas (1992), “Plant biomechanics: Anengineering approach to plant form and function,” The University ofChicago Press, p. 607]. Because of its para-crystalline nature,cellulose makes a disproportionately greater contribution toward tensilestrength of plant tissues than it would if it were amorphous in nature.

[0005] Cell wall of a maize stalk consists mostly of cellulose andhemicellulose, with lignin constituting a minor proportion, i.e., ˜10%(FIG. 1). In a study conducted on three contrasting pairs of hybrids, wehave determined that cellulose concentration in a unit length of stalkbelow the ear is correlated with tensile strength of the stalk (FIG. 2).Stalk lodging is a major problem in maize, accounting for significantyield losses. Increasing cellulose concentration in the wall will resultin a mechanically stronger tissue, reducing the problem of stalklodging.

[0006] The rate of cellulose synthesis exerts major control on theformation of the rest of the wall, as cellulose is its dominantconstituent (FIG. 1). Formation of UDP-glucose, the substrate forcellulose synthase (CesA), occurs through two pathways in plants: onethrough UDP-glucose pyrophosphorylase (UGPase) and the other throughsucrose synthase (FIG. 3). Sucrose synthase (SuSy) catalyzes thereversible reaction:

[0007] Sucrose+UDP⇄UDP−Glucose+Fructose

[0008] Thus, the cleavage reaction provides the precursor for synthesisof starch and cellulose. SuSy uses the energy of the glycosidic bondfrom sucrose to make UDP-glucose from UDP, releasing fructose in theprocess; fructose can then be channeled into UDP-glucose by the UGPasepathway (FIG. 3). While sucrose synthase has historically beenconsidered active in the cytoplasm of plant cells, Amor et al. foundtight association of about half of the total cellular SuSy with theplasma membrane in cotton and suggested that SuSy might channelsubstrate directly from sucrose to CesA [Amor, et al., (1995), “Amembrane-associated form of sucrose synthase and its potential role insynthesis of cellulose and callose in plants,” Proc. Natl. Acad. Sci.USA 92:9353-9357]. Therefore, in a sink tissue, such as growing cornstalk, sucrose synthase provides an economical route for the formationof UDP-glucose from sucrose. In contrast, the UGPase pathway utilizesmore energy in the form of nucleotide triphosphates to produceUDP-glucose from hexose sugars.

[0009] Until the present invention, only two sucrose synthase genes havebeen known in maize, shrunken-1 (Sh1) and constitutive sucrose synthase(Sus1), both of which map to chromosome 9 [Huang, et al. (1994),“Complete nucleotide sequence of the maize (Zea mays L.) sucrosesynthase 2 cDNA,” Plant Physiology Rockville 104:293-294; McCarty, etal. (1986), “The cloning, genetic mapping and expression of theconstitutive sucrose synthase locus of maize,” Proc. Natl. Acad. Sci.USA 83:9099-9103; Werr, et al. (1985), “Structure of the sucrosesynthase (EC 2.4.1.13) gene on chromosome 9 of Zea mays,” EMBO J.4:1373-1380]. These paralogs encode the sucrose synthase isozymes SS1and SS2, respectively.

[0010] Membrane-associated SuSy has also been found in carrot and maize[Carlson, et al. (1996), “Evidence for plasma membrane-associated formsof sucrose synthase in maize,” Molecular and General Genetics252:303-310; Sturm, et al. (1999), “Tissue-specific expression of twogenes for sucrose synthase in carrot (daucus carota L.),” PlantMolecular Biology 39:349-360]. Both the known forms of SuSy in maizewere found to be associated with the plasma membrane fraction fromdeveloping endosperm. Interestingly, Sh1 was suggested to play a greaterrole in cell wall formation than the constitutive sucrose synthase(Sus1), which was purported to contribute more toward starch formation[Chourey, et al. (1998), “Genetic evidence that the two isozymes ofsucrose synthase present in developing maize endosperm are critical, onefor cell integrity and the other for starch biosyntheses,” Molecular andGeneral Genetics 259:88-96]. SuSy is known to become reversiblyphosphorylated at a unique seryl residue [Huber, et al. (1996),“Phosphorylation of serine-15 of maize leaf sucrose synthase,” PlantPhysiology Rockville 112:793-802]. The unphosphorylated form, because ofits relatively greater surface hydrophobicity, is favored to bind themembrane [Winter, et al. (1997), “Membrane association of sucrosesynthase: Changes during the graviresponse and possible control byprotein phosphorylation,” FEBS Letters 420:151-155].

[0011] Sucrose synthase has been suggested to channel substrate to thematrix polysaccharide synthases, based on association with Golgi and aprevious report of its involvement in cellulose synthesis [Buckeridge,et al., (1999), “The mechanism of synthesis of a mixed-linkage(1fwdarw3), (1fwdarw4) beta-D-glucan in maize. Evidence for multiplesites of glucosyl transfer in the synthase complex,”Plant-Physiology-Rockville 120:1105-1116 ]. Direct evidence for thecontribution of SuSy toward substrate generation for cellulose synthesiswas provided by Nakai et al [Nakai, et al. (1999), “Enhancement ofcellulose production by expression of sucrose synthase in Acetobacterxylinum,” Proc. Natl. Acad. Sci. USA 96:14-18]. They obtained a higherlevel of cellulose production in Acetobacter xylinum upon expression ofmung bean sucrose synthase. This bacterium lacks sucrose synthase so islimited to only the UGPase branch of the pathway for making UDP-glucose(FIG. 3). Expression of sucrose synthase also led to a higher level ofUDP-glucose and a lower level of UDP in the bacterium, as would beexpected based on the pathway in FIG. 3.

[0012] Down-regulation of SuSy by antisense approach in carrot reducedthe growth rate [Tang, et al. (1999), “Antisense repression of sucrosesynthase in carrot (Daucus carota L.) affects growth rather than sucrosepartitioning,” Plant-Molecular-Biology 41:465-479]. Levels ofUDP-glucose and cellulose were reduced in the sink tissues in comparisonto the wild type plants, again implying a role for SuSy in substrateproduction for cellulose synthesis. In work with the TUSC (Trait UtilitySystem for Corn; see U.S. Pat. No. 5,962,764, incorporated herein byreference) SuSy mutant, knocking out the constitutive sucrose synthaseled to a reduced cellulose concentration in the walls, as well asreduced amount of total cell wall (Example 8).

[0013] Formation of UDP-glucose from sucrose requires half as muchenergy as if it were to be made from hexose sugars (FIG. 3). Not evenaccounting for the channeling effect, as suggested by Amor et al. [1995,supra], involvement of sucrose synthase in providing substrate tocellulose synthase would lead to improved productivity, particularlyunder stressful conditions, as the energy conserved by this pathwaycould be used for other cellular processes. Over expression of sucrosesynthase under the control of a stalk-preferred promoter in plants couldlead to a greater synthesis of cellulose, thereby strengthening thestalk. Therefore, there is a need in the art for sucrose synthases thatcan be over-expressed under these conditions.

[0014] Sucrose phosphate synthase may participate in UDP-glucosemetabolism, but its role appears to be more to dissipate energy in thesink tissues than to economize the use of sugars (FIG. 3). For example,assuming that all the fructose-6-phosphate and UDP-glucose are derivedfrom the SuSy pathway, at least one ATP is consumed to make sucrose fromthese two substrates only for the former to be cycled through SuSyagain. On the other extreme, i.e., when all the UDP-glucose andfructose-6-phosphate are derived from hexose sugars, formation ofsucrose by sucrose phosphate synthase utilizes 3 NTP per sucrosemolecule produced, two to form UDP-glucose from a hexose sugar and 1 tophosphorylate fructose. In other words, involvement of sucrose phosphatesynthase would consume an extra 1-3 NTP per molecule of sucrose to beincorporated into cellulose, which means a consumption of 3-5 net NTPfor this process.

[0015] Four NTP would be needed per sucrose molecule for its completeconversion to UDP-glucose even if all the sucrose were first to becleaved by invertases, and hexoses were the only sugars available. Eveninvertases dissipate (waste) the energy of the glycosidic bond which isotherwise used by sucrose synthase to form UDP-glucose from UDP. Sucrosephosphate synthase may, however, be important in mediating the formationof sucrose from excess hexoses for transport to other sinks, such asdeveloping ear. This could be important after the deposition ofcellulose into the walls of stalk tissue has slowed down.

[0016] Each hexose sugar molecule, upon complete breakdown byglycolysis, citric acid cycle, and oxidative phosphorylation, produces36 ATP equivalents of energy. As discussed above, each hexose uponactivation into UDP-glucose uses 1 ATP if carried through the SuSypathway and 2 ATP if through the UGPase pathway (FIG. 3). The fractionof sugar utilized, assuming all other processes to be constant, insupporting this conversion is: $\frac{p + {2q}}{36}$

[0017] where p is the proportion of substrates produced by the action ofSuSy; q represents substrates produced from hexose sugars; and p+q=1. Ifall the UDP-glucose were to be derived from the SuSy-mediated pathway,then 2.8% of the sugar would be utilized in producing energy to supportthis reaction. If, on the other hand, hexose was the starting point forall the UDP-glucose produced, then 5.6% of the sugar would be utilizedin generating energy for this series of reactions.

[0018] Routing of any proportion, n, of the sugars through the sucrosephosphate synthase pathway would reduce the efficiency further still asthe NTP utilized for this cycling would be in addition to the ones usedin making UDP-glucose from sucrose or hexose. The following expressionprovides an estimate of the reduction in efficiency:$\frac{\left( {p + {2q}} \right) + {n\left( {p + {3q}} \right)}}{36}$

[0019] If 50% of the sugar is cycled through the sucrose phosphatesynthase pathway and the substrates for this enzyme are derived in equalproportion (i.e., p=q=0.5) from the SuSy and UGPase pathways then,without including the energy needed for the sucrose phosphate synthasepathway to operate, this would translate into 4.2% of the sugarconverted into cellulose being utilized for energy generation to supportthis process. If, however, the energy utilized by the sucrose phosphatesynthase pathway, based on above assumptions, is taken into account,then this number increases to ˜7%, a full 70% extra energy than if nosugar were cycled through this pathway. That is equivalent to burningnearly 3 extra bushels of sugar for every 100 bushels converted intopolysaccharides.

[0020] Thus, the production of cellulose through the sucrose synthasepathway is the most economical means available to plants. One of skillin the art would know of the involvement of sucrose synthase incellulose formation in plants. However, the present invention teachesthat this enzyme is important in supplying substrate for cellulosesynthesis (Example 8).

[0021] As stalk composition contributes to numerous quality factorsimportant in maize breeding, what is needed in the art are products andmethods for manipulating cellulose concentration in the cell wall andthereby altering plant stalk quality to provide, for example, increasedstandability. It would be desirable to over-express sucrose synthase,preferably under the control of a stalk-preferred promoter, to improvestalk strength in maize.

[0022] Another attribute of importance is grain handling ability, i.e.,reducing grain breakage during combining, transport, and movement intostorage. Grain strength in cereals such as wheat and barley is mainlyderived from the pericarp, which allows for a softer endosperm. It wouldbe desirable to increase cellulose in the pericarp by over-expressingsucrose synthase under the control of a pericarp-specific promoter.

[0023] The present invention provides these and other advantages.

SUMMARY OF THE INVENTION

[0024] We have identified a heretofore unknown cDNA for a third sucrosesynthase gene, Sus3, from a proprietary genome database (see ZmSus3,Examples 9 and 10). Sus3 maps to the short arm of chromosome 1 (bin1.04). The ESTs for this gene are found in a variety of tissues, albeitat a much lower frequency than those for Sus1, indicating that thisgene, like Sus1, is expressed constitutively.

[0025] Generally, it is the object of the present invention to providenucleic acids and proteins relating to sucrose synthase 3 (Sus3). It isan object of the present invention to provide transgenic plantscomprising the nucleic acids of the present invention, and methods formodulating, in a transgenic plant, expression of the nucleic acids ofthe present invention. More specifically, it is also an object of thepresent invention to manipulate cellulose concentration in the cell walland to alter grain quality and/or plant stalk quality. It is anotherobject of the present invention to alter expression of sucrose synthasein a plant to improve stalk quality and/or stalk strength. It is anotherobject of this invention to alter expression of sucrose synthase in aplant to improve grain quality and/or grain strength.

[0026] Therefore, in one aspect the present invention relates to anisolated nucleic acid comprising a member selected from the groupconsisting of (a) a polynucleotide having a specified sequence identityto a polynucleotide of the present invention; (b) a polynucleotide whichis complementary to the polynucleotide of (a); and, (c) a polynucleotidecomprising a specified number of contiguous nucleotides from apolynucleotide of (a) or (b). The isolated nucleic acid can be DNA.

[0027] In other aspects the present invention relates to: 1) recombinantexpression cassettes, comprising a nucleic acid of the present inventionoperably linked to a promoter, 2) a host cell into which has beenintroduced the recombinant expression cassette, and 3) a transgenicplant comprising the recombinant expression cassette. The host cell andplant are optionally from maize, wheat, rice, or soybean.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIG. 1. Circle graph depicting chemical composition of corn stalk.Two internodes, 2nd and 3rd below the ear, were harvested 60 days afterflowering, dried and ground. Structural dry matter was determined bywashing the powdered material with buffer followed bymethanol:chloroform. Cellulose was determined gravimetrically byUpdegraff's method by boiling the ground material or structural drymatter in acetic acid-nitric acid mix, and lignin by Klason method. Ashwas determined by incinerating the samples in a 600° C. oven for 4hours. Protein was assumed to be around 3%. Soluble component wasderived by subtracting structural dry matter from total dry matter.Hemicellulose concentration was estimated by subtracting cellulose,lignin, protein, and ash from structural dry matter. Mature monocotwalls are known to have very little pectin.

[0029]FIG. 2. Bar graph depicting amount of cellulose in corn hybrids.Two internodes, 2nd and 3rd below the ear, were harvested 60 days afterflowering, dried and ground. Structural dry matter was determined bywashing the powdered material with buffer followed bymethanol:chloroform. Cellulose was determined gravimetrically byUpdegraff's method by boiling the ground material or structural drymatter in acetic acid-nitric acid mix, and lignin by Klason method. Ashwas determined by incinerating the samples in a 600° C. oven for 4hours. Protein was assumed to be around 3%. Soluble component wasderived by subtracting structural dry matter from total dry matter.Hemicellulose concentration was estimated by subtracting cellulose,lignin, protein, and ash from structural dry matter. Mature monocotwalls are known to have very little pectin.

[0030]FIG. 3. Schematic representation of partial pathways for synthesisof UDP-glucose. Abbreviations: ATP, adenosine triphosphate; CesA,cellulose synthase; HK, hexokinase; PPase, pyrophosphatase; PPi,pyrophosphate; SPP, sucrose phosphate phosphatase; SPS, sucrosephosphate synthase; SuSy, sucrose synthase; UDPG or UDP-Glucose, uridinediphosphate glucose; UGPase, UDPG pyrophosphorylase; UTP, uridinetriphosphate.

[0031]FIG. 4. Circle graph depicting distribution of Sus1 ESTs in maizetissues. For Sus1, 230 ESTs were found in the genome database consistingof approximately 400,000 total ESTs.

[0032]FIG. 5. Circle graph depicting distribution of Sus3 ESTs in maizetissues. Out of approximately 400,000 ESTs in the genome database, 26ESTs were found for Sus3.

[0033]FIG. 6. In this representation of the genomic clone of ZmSus1,narrow bars represent introns and wider bars represent exons.Approximate location of the two independent Mu-insertional alleles isshown by the down arrows in the 12^(th) exon (exact location is in thesignature sequences shown above).

[0034]FIG. 7. Table of data from analysis of cellulose and cell wallcontent in Sus1 mutants.

[0035]FIG. 8. Multiple alignment of maize sucrose synthase amino acidsequences.

[0036]FIG. 9. Multiple alignment of maize sucrose synthasepolynucleotides.

[0037]FIG. 10. Sequence of SEQ ID NO: 13, Sorghum EST having GenBankAccession No. BF481989. The ATG encoding the first methionine in theopen reading frame of SEQ ID NO: 11 is shown in bold. The sequenceutilized to provide the deduced full length Sus3 sequence is underlined.

[0038]FIG. 11. The combination of maize and sorghum Sus3 sequences usedto create SEQ ID NO: 11. The portions of sorghum sequence selected fromSEQ ID NO: 13 and the selected maize sequence selected from SEQ ID NO: 1are shown separately. Before combining the sequence from SEQ ID NO: 1with the shown sorghum sequence from SEQ ID NO: 13 to create SEQ ID NO:11, the nucleotides in SEQ ID NO 1 shown as highlighted withstrikethrough should be removed removed.

DETAILED DESCRIPTION OF THE INVENTION Overview

[0039] A. Nucleic Acids and Protein of the Present Invention

[0040] The polynucleotide sequences of SEQ ID. NOS. 1 and 11, andpolypeptide sequences of SEQ ID. NOS. 2 and 12, represent apolynucleotide and polypeptide of the present invention. A nucleic acidof the present invention comprises a polynucleotide of the presentinvention. A protein of the present invention comprises a polypeptide ofthe present invention.

[0041] B. Exemplary Utilities of the Present Invention

[0042] The present invention provides utility in such exemplaryapplications as manipulating cellulose concentration in the cell walland thereby altering plant stalk quality to provide, for example,increased standability. It would be desirable to over-express sucrosesynthase, preferably under the control of a stalk-preferred promoter, toimprove plant strength in maize.

[0043] Another attribute of importance is grain handling ability, i.e.,reducing grain breakage during combining, transport, and movement intostorage. Grain strength in cereals such as wheat and barley is mainlyderived from the pericarp, which allows for a softer endosperm. It wouldbe desirable to increase cellulose in the pericarp by over-expressingsucrose synthase, preferably under the control of a pericarp-preferredpromoter.

[0044] C. Exemplary Preferable Embodiments

[0045] While the various preferred embodiments are disclosed throughoutthe specification, exemplary preferable embodiments include thefollowing:

[0046] (i) Expression pattern of sucrose synthase genes. Sus1 isrepresented by approximately 230 and Sus3 by about 26 ESTs found inPioneer Hi-Bred International, Inc. proprietary genome databases, whichinclude data from numerous proprietary nucleic acid librariesrepresenting plant tissues at a variety of developmental stages. TheseEST findings act as a sort of electronic Northern and provide evidencethat Sus3 is expressed at a much lower level than Sus1 (FIGS. 4 and 5).Both Sus1 and Sus3 are expressed in a variety of tissues and thereforecan be classified as constitutive sucrose synthases. However, Sus3appears to be somewhat preferentially expressed in the kernel, where 50%of its ESTs are found. In comparison, only about 10% of ESTs (15% whenthe callus tissue is excluded) for Sus1 are found in the kernel tissue.One striking difference is that Sus3 does not seem to be expressed inthe callus tissue at all, whereas about half of the ESTs for Sus1 arefound in libraries derived from this tissue.

[0047] (ii) Promoters. Preferred promoters include but are not limitedto: the Actin-1 promoter from rice (McElroy et al. 1990, Plant Cell2:163-171); the rice tungro bacilliform virus promoter (Yin et al. 1995,Plant Journal 7(6): 969-980); the Agrobacterium rhizogenes ROIC promoterand the maize Sh promoter (Graham et al. 1997, Plant Molecular Biology33:729-735); the tissue-preferred promoter described in U.S. Pat. No.5,986,174, herein incorporated by reference; S2A promoter from maize oralfalfa (Abrahams et al. 1995, Plant Molecular Biology 27(3):513-528);maize Adh2 promoter elements (Paul et al. 1994, Plant Journal5(4):523-533); CoYMV promoter (Medberry et al. 1992, Plant Cell 4(2):185-192); bean grp 1.8 promoter and regulatory elements therein (Kelleret al. 1994, Plant Molecular Biology 26(2):747-756); tomato prosysteminpromoter (Jacinto et al. 1997, Planta 203(4):406-412); maize gene Hrgppromoter (Menossi et al. 1997, Plant Science 125 (2):189-200); maizeSus1 promoter (Huang, X. et al. (1998) Euphytica 103(1):17-21); promoterof maize gene P-rr (Sidorenko et al. 1999, Plant Molecular Biology,39:11-19), and maize promoter mZE40-2 described in U.S. patentapplication Ser. No. 09/666,179.

Definitions

[0048] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range. Amino acidsmay be referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBMBNomenclature Commission. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes. Unless otherwise providedfor, software, electrical, and electronics terms as used herein are asdefined in The New IEEE Standard Dictionary of Electrical andElectronics Terms (5^(th) edition, 1993). The terms defined below aremore fully defined by reference to the specification as a whole. Sectionheadings provided throughout the specification are not limitations tothe various objects and embodiments of the present invention.

[0049] By “amplified” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Cangene, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

[0050] As used herein, “antisense orientation” includes reference to aduplex polynucleotide sequence that is operably linked to a promoter inan orientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

[0051] The terms “alter” or “modify” or “modulate”, with respect toexpression of nucleic acids or proteins, include reference to methods ofup-regulation and down-regulation. Up-regulation may be achieved, forexample, through increased transcription and/or translation of a gene ofinterest, through means such as operably linking the gene of interest toa promoter sequence which favors increased transcription; through addingor over-expressing a necessary substrate in a metabolic pathway; throughthe blocking of antagonistic molecules; or by other means known to oneof skill in the art. Down-regulation may be achieved, for example,through antisense technology (see, e.g., Sheehy et al., Proc. Nat'l.Acad. Sci. (USA) 85: 8805-8809 (1988); and Shewmaker, Hiatt, et al.,U.S. Pat. No. 5,759,829); through RNA interference (see Napoli et al.,The Plant Cell 2: 279-289 (1990); U.S. Pat. No. 5,034,323; Sharp, Genes& Development 13:139-141(1999); Zamore et al., Cell 101:25-33 (2000);Montgomery et al., PNAS USA 95:15502-15507 (1998); virus-induced genesilencing (Burton, et al., The Plant Cell 12:691-705 (2000); Baulcombe,Curr. Opn. Plant Bio. 2:109-113 (1999)); through the use oftarget-RNA-specific ribozymes (Haseloff et al., Nature 334: 585-591(1988)); through hairpin-loop suppression (Smith et al., Nature407:319-320 (2000)); and through other methods known to those of skillin the art. Said up- or down-regulation may be directed preferentially,such as within certain tissues, under particular environmentalconditions, and/or at certain stages of plant development.

[0052] By “encoding” or “encoded”, with respect to a specified nucleicacid, is meant comprising the information for translation into thespecified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code. However,variants of the universal code, such as are present in some plant,animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, orthe ciliate Macronucleus, may be used when the nucleic acid is expressedtherein.

[0053] When the nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed. For example, although nucleicacid sequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498(1989)). Thus, the maize preferred codon for a particular amino acid maybe derived from known gene sequences from maize. Maize codon usage for28 genes from maize plants is listed in Table 4 of Murray et al., supra.

[0054] As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (non-synthetic), endogenous, biologically (e.g.,structurally or catalytically) active form of the specified protein.Methods to determine whether a sequence is full-length are well known inthe art including such exemplary techniques as northern or westernblots, primer extension, S1 protection, and ribonuclease protection.See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Comparison to known full-lengthhomologous (orthologous and/or paralogous) sequences can also be used toidentify full-length sequences of the present invention. Additionally,consensus sequences typically present at the 5′ and 3′ untranslatedregions of mRNA aid in the identification of a polynucleotide asfull-length. For example, the consensus sequence ANNNNAUGG, where theunderlined codon represents the N-terminal methionine, aids indetermining whether the polynucleotide has a complete 5′ end. Consensussequences at the 3′ end, such as polyadenylation sequences, aid indetermining whether the polynucleotide has a complete 3′ end.

[0055] As used herein, “heterologous” in reference to a nucleic acid isa nucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by human intervention. For example, apromoter operably linked to a heterologous structural gene is from aspecies different from that from which the structural gene was derived,or, if from the same species, one or both are substantially modifiedfrom their original form. A heterologous protein may originate from aforeign species or, if from the same species, is substantially modifiedfrom its original form by human intervention.

[0056] By “host cell” is meant a cell which contains a vector andsupports the replication and/or expression of the vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells. A particularly preferredmonocotyledonous host cell is a maize host cell.

[0057] The term “introduced” includes reference to the incorporation ofa nucleic acid into a eukaryotic or prokaryotic cell where the nucleicacid may be incorporated into the genome of the cell (e.g., chromosome,plasmid, plastid or mitochondrial DNA), converted into an autonomousreplicon, or transiently expressed (e.g., transfected mRNA). The termincludes such nucleic acid introduction means as “transfection”,“transformation” and “transduction”.

[0058] The term “isolated” refers to material, such as a nucleic acid ora protein, which is substantially free from components that normallyaccompany or interact with it as found in its naturally occurringenvironment. The isolated material optionally comprises material notfound with the material in its natural environment, or if the materialis in its natural environment, the material has been synthetically(non-naturally) altered by human intervention to a composition and/orplaced at a location in the cell (e.g., genome or subcellular organelle)not native to a material found in that environment. The alteration toyield the synthetic material can be performed on the material within orremoved from its natural state. For example, a naturally occurringnucleic acid becomes an isolated nucleic acid if it is altered, or if itis transcribed from DNA which has been altered, by means of humanintervention performed within the cell from which it originates. See,e.g., Compounds and Methods for Site Directed Mutagenesis in EukaryoticCells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous SequenceTargeting in Eukaryotic Cells; Zarling et al., WO 93/22443. Likewise, anaturally occurring nucleic acid (e.g., a promoter) becomes isolated ifit is introduced by non-naturally occurring means to a locus of thegenome not native to that nucleic acid. Nucleic acids which are“isolated” as defined herein, are also referred to as “heterologous”nucleic acids.

[0059] As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer, or chimeras thereof, ineither single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids).

[0060] By “nucleic acid library” is meant a collection of isolated DNAor RNA molecules which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism, tissue, or ofa cell type from that organism. Construction of exemplary nucleic acidlibraries, such as genomic and cDNA libraries, is taught in standardmolecular biology references such as Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology, Vol. 152, AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al., MolecularCloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., Eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994).

[0061] As used herein “operably linked” includes reference to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0062] As used herein, the term “plant” includes reference to wholeplants and their progeny; plant cells; plant parts or organs, such asembryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, leaves,husks, stalks, stems, roots, root tips, anthers, silk and the like.Plant cell, as used herein, further includes, without limitation, cellsobtained from or found in: seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. Plant cells can alsobe understood to include modified cells, such as protoplasts, obtainedfrom the aforementioned tissues. The class of plants which can be usedin the methods of the invention is generally as broad as the class ofhigher plants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. A particularly preferredplant is Zea mays.

[0063] As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or chimeras or analogsthereof that have the essential nature of a natural deoxy- orribo-nucleotide in that they hybridize, under stringent hybridizationconditions, to substantially the same nucleotide sequence as naturallyoccurring nucleotides and/or allow translation into the same aminoacid(s) as the naturally occurring nucleotide(s). A polynucleotide canbe full-length or a subsequence of a native or heterologous structuralor regulatory gene. Unless otherwise indicated, the term includesreference to the specified sequence as well as the complementarysequence thereof. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritylated bases, to name justtwo examples, are polynucleotides as the term is used herein. It will beappreciated that a great variety of modifications have been made to DNAand RNA that serve many useful purposes known to those of skill in theart. The term polynucleotide as it is employed herein embraces suchchemically, enzymatically or metabolically modified forms ofpolynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

[0064] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Further, this invention contemplatesthe use of both the methionine-containing and the methionine-less aminoterminal variants of the protein of the invention.

[0065] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells whether or not its origin is a plant cell. Exemplary plantpromoters include, but are not limited to, those that are obtained fromplants, plant viruses, and bacteria, which comprise genes expressed inplant cells such as Agrobacterium or Rhizobium. Examples of promotersunder developmental control include promoters that preferentiallyinitiate transcription in certain tissues, such as leaves, roots, orseeds. Such promoters are referred to as “tissue preferred”. Promoterswhich initiate transcription only, or almost only, in certain tissue arereferred to as “tissue specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. A promoter may havespatial or temporal specificity, capable of initiating transcriptionpreferentially with respect to conditions of space or time. An“inducible” or “repressible” promoter is a promoter which is underenvironmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterwhich is active under most environmental conditions.

[0066] As used herein “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed,under-expressed or not expressed at all as a result of humanintervention. The term “recombinant” as used herein does not encompassthe alteration of the cell or vector by naturally occurring events(e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout human intervention.

[0067] As used herein, a “recombinant expression cassette” is a nucleicacid construct, generated recombinantly or synthetically, with a seriesof specified nucleic acid elements which permit transcription of aparticular nucleic acid in a host cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

[0068] The term “residue” or “amino acid residue” or “amino acid” areused interchangeably herein to refer to an amino acid that isincorporated into a protein, polypeptide, or peptide (collectively“protein”). The amino acid may be a naturally occurring amino acid and,unless otherwise limited, may encompass non-natural analogs of naturalamino acids that can function in a similar manner as naturally occurringamino acids.

[0069] The term “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other.

[0070] The term “stringent conditions” or “stringent hybridizationconditions” includes reference to conditions under which a probe willselectively hybridize to its target sequence, to a detectably greaterdegree than to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, optionally less than 500 nucleotides inlength.

[0071] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1 % SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

[0072] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridizationand/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. Hybridizationand/or wash conditions can be applied for at least 10, 30, 60, 90, 120,or 240 minutes. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

[0073] As used herein, “transgenic plant” includes reference to a plantwhich comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant expression cassette.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. The term “transgenic”as used herein does not encompass the alteration of the genome(chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

[0074] As used herein, “vector” includes reference to a nucleic acidused in introduction of a polynucleotide of the present invention into ahost cell. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

[0075] The following terms are used to describe the sequencerelationships between a polynucleotide/polypeptide of the presentinvention and a reference polynucleotide/polypeptide: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, and (d)“percentage of sequence identity”.

[0076] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison with apolynucleotide/polypeptide of the present invention. A referencesequence may be a subset or the entirety of a specified sequence; forexample, as a segment of a full-length cDNA or gene sequence, or thecomplete cDNA or gene sequence.

[0077] (b) As used herein, “comparison window” includes reference to acontiguous and specified segment of a polynucleotide/polypeptidesequence, wherein the polynucleotide/polypeptide sequence may becompared to a reference sequence and wherein the portion of thepolynucleotide/polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Generally, the comparison window is atleast 20 contiguous nucleotides/amino acid residues in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide/polypeptide sequence, a gappenalty is typically introduced and is subtracted from the number ofmatches.

[0078] Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2: 482 (1981); by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970); by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85: 2444(1988); by computerized implementations of these algorithms, including,but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics,Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994).

[0079] The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995);Altschul et al., J. Mol. Biol., 215:403-410 (1990); and, Altschul etal., Nucleic Acids Res. 25:3389-3402 (1997).

[0080] Software for performing BLAST analyses is publicly available,e.g., through the National Center for Biotechnology Information. Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold. These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

[0081] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

[0082] BLAST searches assume that proteins can be modeled as randomsequences. However, many real proteins comprise regions of nonrandomsequences which may be homopolymeric tracts, short-period repeats, orregions enriched in one or more amino acids. Such low-complexity regionsmay be aligned between unrelated proteins even though other regions ofthe protein are entirely dissimilar. A number of low-complexity filterprograms can be employed to reduce such low-complexity alignments. Forexample, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993))and XNU (Claverie and States, Comput Chem., 17:191-201 (1993))low-complexity filters can be employed alone or in combination.

[0083] Unless otherwise stated, nucleotide and proteinidentity/similarity values provided herein are calculated using GAP (GCGVersion 10) under default values.

[0084] GAP (Global Alignment Program) can also be used to compare apolynucleotide or polypeptide of the present invention with a referencesequence. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48: 443-453,1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 100. Thus, for example, the gapcreation and gap extension penalties can each independently be: 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater.

[0085] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0086] Multiple alignment of the sequences can be performed using theCLUSTAL method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments using the CLUSTAL method areKTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0087] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences includes referenceto the residues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

[0088] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Utilities

[0089] The present invention provides, among other things, compositionsand methods for altering or modulating the level of polynucleotides andpolypeptides of the present invention in plants. In particular, thepolynucleotides and polypeptides of the present invention can beexpressed temporally or spatially, e.g., at developmental stages, intissues, and/or in quantities, which are uncharacteristic ofnon-recombinantly engineered plants.

[0090] The present invention also provides isolated nucleic acidscomprising polynucleotides of sufficient length and complementarity to apolynucleotide of the present invention to use as probes oramplification primers in the detection, quantitation, or isolation ofgene transcripts. For example, isolated nucleic acids of the presentinvention can be used as probes in detecting deficiencies in the levelof mRNA in screenings for desired transgenic plants, for detectingmutations in the gene (e.g., substitutions, deletions, or additions),for monitoring up-regulation of expression or changes in enzyme activityin screening assays of compounds, for detection of any number of allelicvariants (polymorphisms), orthologs, or paralogs of the gene, or forsite directed mutagenesis in eukaryotic cells (see, e.g., U.S. Pat. No.5,565,350). The isolated nucleic acids of the present invention can alsobe used for recombinant expression of their encoded polypeptides, or foruse as immunogens in the preparation and/or screening of antibodies. Theisolated nucleic acids of the present invention can also be employed foruse in sense or antisense suppression of one or more genes of thepresent invention in a host cell, tissue, or plant. Aftachment ofchemical agents which bind, intercalate, cleave and/or cross-link to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation.

[0091] The present invention also provides isolated proteins comprisinga polypeptide of the present invention (e.g., preproenzyme, proenzyme,or enzymes). The present invention also provides proteins comprising atleast one epitope from a polypeptide of the present invention. Theproteins of the present invention can be employed in assays for enzymeagonists or antagonists of enzyme function, or for use as immunogens orantigens to obtain antibodies specifically immunoreactive with a proteinof the present invention. Such antibodies can be used in assays forexpression levels, for identifying and/or isolating nucleic acids of thepresent invention from expression libraries, for identification ofhomologous polypeptides from other species, or for purification ofpolypeptides of the present invention.

[0092] The isolated nucleic acids and polypeptides of the presentinvention can be used over a broad range of plant types, particularlymonocots such as the species of the family Gramineae including Hordeum,Secale, Oryza, Triticum, Sorghum (e.g., S. bicolor) and Zea (e.g., Z.mays), and dicots such as Glycine.

[0093] The isolated nucleic acid and proteins of the present inventioncan also be used in species from the genera: Cucurbita, Rosa, Vitis,Juglans, Fragaria, Lotus, Medica go, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browallia, Pisum, Phaseolus, Lolium, and Avena.

Nucleic Acids

[0094] The present invention provides, among other things, isolatednucleic acids of RNA, DNA, and analogs and/or chimeras thereof,comprising a polynucleotide of the present invention.

[0095] A polynucleotide of the present invention is inclusive of:

[0096] (a) an isolated polynucleotide encoding SEQ ID NO: 2 or SEQ IDNO:12, including exemplary polynucleotides of the present invention;

[0097] (b) an isolated polynucleotide which is the product ofamplification from a plant nucleic acid library using primer pairs whichselectively hybridize under stringent conditions to loci within apolynucleotide of the present invention;

[0098] (c) an isolated polynucleotide which selectively hybridizes to apolynucleotide of (a) or (b);

[0099] (d) an isolated polynucleotide having a specified sequenceidentity with polynucleotides of (a), (b), or (c);

[0100] (e) an isolated polynucleotide encoding a protein having aspecified number of contiguous amino acids from a prototype polypeptide,wherein the protein is specifically recognized by antisera elicited bypresentation of the protein and wherein the protein does not detectablyimmunoreact to antisera which have been fully immunosorbed with theprotein;

[0101] (f) complementary sequences of polynucleotides of (a), (b), (d),or (e); and

[0102] (g) an isolated polynucleotide comprising at least a specificnumber of contiguous nucleotides from a polynucleotide of (a), (b), (c),(d), (e), or (f);

[0103] (h) an isolated polynucleotide from a full-length enriched cDNAlibrary having the physico-chemical property of selectively hybridizingto a polynucleotide of (a), (b), (c), (d), (e), (f), or (g);

[0104] (i) an isolated polynucleotide made by the process of: 1)providing a full-length enriched nucleic acid library, 2) selectivelyhybridizing the polynucleotide to a polynucleotide of (a), (b), (c),(d), (e), (f), (g), or (h), thereby isolating the polynucleotide fromthe nucleic acid library.

[0105] A. Polynucleotides Encoding A Polypeptide of the PresentInvention

[0106] As indicated in (a), above, the present invention providesisolated nucleic acids comprising a polynucleotide of the presentinvention, wherein the polynucleotide encodes a polypeptide of thepresent invention. Every nucleic acid sequence herein that encodes apolypeptide also, by reference to the genetic code, describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine; and UGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Thus, each silent variation of a nucleic acid whichencodes a polypeptide of the present invention is implicit in eachdescribed polypeptide sequence and is within the scope of the presentinvention. Accordingly, the present invention includes polynucleotidesof the present invention and polynucleotides encoding a polypeptide ofthe present invention.

[0107] B. Polynucleotides Amplified from a Plant Nucleic Acid Library

[0108] As indicated in (b), above, the present invention provides anisolated nucleic acid comprising a polynucleotide of the presentinvention, wherein the polynucleotides are amplified, under nucleic acidamplification conditions, from a plant nucleic acid library. Nucleicacid amplification conditions for each of the variety of amplificationmethods are well known to those of ordinary skill in the art. The plantnucleic acid library can be constructed from a monocot such as a cerealcrop. Exemplary cereals include corn, sorghum, alfalfa, canola, wheat,and rice. The plant nucleic acid library can also be constructed from adicot such as soybean. Zea mays lines B73, A632, BMS, W23, and Mo17 areknown and publicly available. Other publicly known and available maizelines can be obtained from the Maize Genetics Cooperation (Urbana,Ill.). Wheat lines are available from the Wheat Genetics Resource Center(Manhattan, Kans.).

[0109] The nucleic acid library may be a cDNA library, a genomiclibrary, or a library generally constructed from nuclear transcripts atany stage of intron processing. cDNA libraries can be normalized toincrease the representation of relatively rare cDNAs. In optionalembodiments, the cDNA library is constructed using an enrichedfull-length cDNA synthesis method. Examples of such methods includeOligo-Capping (Maruyama, K. and Sugano, S. Gene 138: 171-174,1994),Biotinylated CAP Trapper (Carninci, et al. Genomics 37: 327-336,1996),and CAP Retention Procedure (Edery, E., Chu, L. L., et al. Molecular andCellular Biology 15: 3363-3371, 1995). Rapidly growing tissues orrapidly dividing cells are preferred for use as an mRNA source forconstruction of a cDNA library. Growth stages of corn are described in“How a Corn Plant Develops,” Special Report No. 48, Iowa StateUniversity of Science and Technology Cooperative Extension Service,Ames, Iowa, February 1993;http://www.ag.iastate.edu/departments/agronomy/comtitle.html.

[0110] A polynucleotide of this embodiment (or subsequences thereof) canbe obtained, for example, by using amplification primers which areselectively hybridized and primer extended, under nucleic acidamplification conditions, to at least two sites within a polynucleotideof the present invention, or to two sites within the nucleic acid whichflank and comprise a polynucleotide of the present invention, or to asite within a polynucleotide of the present invention and a site withinthe nucleic acid which comprises it. Methods for obtaining 5′ and/or 3′ends of a vector insert are well known in the art. See, e.g., RACE(Rapid Amplification of Complementary Ends) as described in Frohman, M.A., in PCR Protocols: A Guide to Methods and Applications, M. A. Innis,D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (Academic Press, Inc.,San Diego), pp. 28-38 (1990)); see also, U.S. Pat. No. 5,470,722, andCurrent Protocols in Molecular Biology, Unit 15.6, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995); Frohmanand Martin, Techniques 1:165 (1989).

[0111] Optionally, the primers are complementary to a subsequence of thetarget nucleic acid which they amplify but may have a sequence identityranging from about 85% to 99% relative to the polynucleotide sequence towhich they are designed to anneal. As those skilled in the art willappreciate, the sites to which the primer pairs will selectivelyhybridize are chosen such that a single contiguous nucleic acid can beformed under the desired nucleic acid amplification conditions. Theprimer length in nucleotides is selected from the group of integersconsisting of from at least 15 to 50. Thus, the primers can be at least15, 18, 20, 25, 30, 40, or 50 nucleotides in length. Those of skill willrecognize that a lengthened primer sequence can be employed to increasespecificity of binding (i.e., annealing) to a target sequence. Anon-annealing sequence at the 5′end of a primer (a “tail”) can be added,for example, to introduce a cloning site at the terminal ends of theamplicon.

[0112] The amplification products can be translated using expressionsystems well known to those of skill in the art. The resultingtranslation products can be confirmed as polypeptides of the presentinvention by, for example, assaying for the appropriate catalyticactivity (e.g., specific activity and/or substrate specificity), orverifying the presence of one or more epitopes which are specific to apolypeptide of the present invention. Methods for protein synthesis fromPCR derived templates are known in the art and available commercially.See, e.g., Amersham Life Sciences, Inc, Catalog '97, p.354.

[0113] C. Polynucleotides Which Selectively Hybridize to aPolynucleotide of (A) or (B)

[0114] As indicated in (c), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides selectively hybridize, underselective hybridization conditions, to a polynucleotide of sections (A)or (B) as discussed above. Thus, the polynucleotides of this embodimentcan be used for isolating, detecting, and/or quantifying nucleic acidscomprising the polynucleotides of (A) or (B). For example,polynucleotides of the present invention can be used to identify,isolate, or amplify partial or full-length clones in a depositedlibrary. In some embodiments, the polynucleotides are genomic or cDNAsequences isolated or otherwise complementary to a cDNA from a dicot ormonocot nucleic acid library. Exemplary species of monocots and dicotsinclude, but are not limited to: maize, canola, soybean, cotton, wheat,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice.The cDNA library comprises at least 50% to 95% full-length sequences(for example, at least 50%, 60%, 70%, 80%, 90%, or 95% full-lengthsequences). The cDNA libraries can be normalized to increase therepresentation of rare sequences. See, e.g., U.S. Pat. No. 5,482,845.Low stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% to 80% sequence identity and can be employed toidentify orthologous or paralogous sequences.

[0115] D. Polynucleotides Having a Specific Sequence Identity with thePolynucleotides of (A), (B) or (C)

[0116] As indicated in (d), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides have a specified identity at thenucleotide level to a polynucleotide as disclosed in sections (A), (B),or (C), above. Identity can be calculated using, for example, the BLAST,CLUSTALW, or GAP algorithms under default conditions. The percentage ofidentity to a reference sequence is at least 50% and, rounded upwards tothe nearest integer, can be expressed as an integer selected from thegroup of integers consisting of from 50 to 99. Thus, for example, thepercentage of identity to a reference sequence can be at least 60%, 70%,75%, 80%, 85%, 90%, or 95%.

[0117] Optionally, the polynucleotides of this embodiment will encode apolypeptide that will share an epitope with a polypeptide encoded by thepolynucleotides of sections (A), (B), or (C). Thus, thesepolynucleotides encode a first polypeptide which elicits production ofantisera comprising antibodies which are specifically reactive to asecond polypeptide encoded by a polynucleotide of (A), (B), or (C).However, the first polypeptide does not bind to antisera raised againstitself when the antisera have been fully immunosorbed with the firstpolypeptide. Hence, the polynucleotides of this embodiment can be usedto generate antibodies for use in, for example, the screening ofexpression libraries for nucleic acids comprising polynucleotides of(A), (B), or (C), or for purification of, or in immunoassays for,polypeptides encoded by the polynucleotides of (A), (B), or (C). Thepolynucleotides of this embodiment comprise nucleic acid sequences whichcan be employed for selective hybridization to a polynucleotide encodinga polypeptide of the present invention.

[0118] Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT patent publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent publication Nos. 92/05258,92/14843, and 97/20078. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

[0119] E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and Cross-Reactive to the Prototype Polypeptide

[0120] As indicated in (e), above, the present invention providesisolated nucleic acids comprising polynucleotides of the presentinvention, wherein the polynucleotides encode a protein having asubsequence of contiguous amino acids from a prototype polypeptide ofthe present invention such as are provided in (a), above. The length ofcontiguous amino acids from the prototype polypeptide is selected fromthe group of integers consisting of from at least 10 to the number ofamino acids within the prototype sequence. Thus, for example, thepolynucleotide can encode a polypeptide having a subsequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids fromthe prototype polypeptide. Further, the number of such subsequencesencoded by a polynucleotide of the instant embodiment can be any integerselected from the group consisting of from 1 to 20, such as 2, 3, 4, or5. The subsequences can be separated by any integer of nucleotides from1 to the number of nucleotides in the sequence such as at least 5, 10,15, 25, 50, 100, or 200 nucleotides.

[0121] The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such asbut not limited to, a polypeptide encoded by the polynucleotide of (a)or (b), above. Generally, however, a protein encoded by a polynucleotideof this embodiment does not bind to antisera raised against theprototype polypeptide when the antisera have been fully immunosorbedwith the prototype polypeptide. Methods of making and assaying forantibody binding specificity/affinity are well known in the art.Exemplary immunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

[0122] In a preferred assay method, fully immunosorbed and pooledantisera which are elicited to the prototype polypeptide can be used ina competitive binding assay to test the protein. The concentration ofthe prototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants, and minor recombinant modifications to a prototypepolypeptide.

[0123] A polynucleotide of the present invention optionally encodes aprotein having a molecular weight as the non-glycosylated protein within20% of the molecular weight of the full-length non-glycosylatedpolypeptides of the present invention. Molecular weight can be readilydetermined by SDS-PAGE under reducing conditions. Optionally, themolecular weight is within 15% of a full-length polypeptide of thepresent invention, more preferably within 10% or 5%, and most preferablywithin 3%, 2%, or 1% of a full-length polypeptide of the presentinvention.

[0124] Optionally, the polynucleotides of this embodiment will encode aprotein having a specific enzymatic activity at least 50%, 60%, 80%, or90% of a cellular extract comprising the native, endogenous full-lengthpolypeptide of the present invention. Further, the proteins encoded bypolynucleotides of this embodiment will optionally have a substantiallysimilar affinity constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, k_(cat)) as the native endogenous,full-length protein. Those of skill in the art will recognize thatk_(cat)/K_(m) value determines the specificity for competing substratesand is often referred to as the specificity constant. Proteins of thisembodiment can have a k_(cat)/K_(m) value at least 10% of a full-lengthpolypeptide of the present invention as determined using the endogenoussubstrate of that polypeptide. Optionally, the k_(cat)/K_(m) value willbe at least 20%, 30%, 40%, 50%, and most preferably at least 60%, 70%,80%, 90%, or 95% the k_(cat)/K_(m) value of the full-length polypeptideof the present invention. Determination of k_(cat), K_(m), andk_(cat)/K_(m) can be determined by any number of means well known tothose of skill in the art. For example, the initial rates (i.e., thefirst 5% or less of the reaction) can be determined using rapid mixingand sampling techniques (e.g., continuous-flow, stopped-flow, or rapidquenching techniques), flash photolysis, or relaxation methods (e.g.,temperature jumps) in conjunction with such exemplary methods ofmeasuring as spectrophotometry, spectrofluorimetry, nuclear magneticresonance, or radioactive procedures. Kinetic values are convenientlyobtained using a Lineweaver-Burk or Eadie-Hofstee plot.

[0125] F. Polynucleotides Complementary to the Polynucleotides of(A)-(E)

[0126] As indicated in (f), above, the present invention providesisolated nucleic acids comprising polynucleotides complementary to thepolynucleotides of paragraphs A-E, above. As those of skill in the artwill recognize, complementary sequences base-pair throughout theentirety of their length with the polynucleotides of sections (A)-(E)(i.e., have 100% sequence identity over their entire length).Complementary bases associate through hydrogen bonding in doublestranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil.

[0127] G. Polynucleotides Which are Subsequences of the Polynucleotidesof (A)-(F)

[0128] As indicated in (g), above, the present invention providesisolated nucleic acids comprising polynucleotides which comprise atleast 15 contiguous bases from the polynucleotides of sections (A)through (F) as discussed above. The length of the polynucleotide isgiven as an integer selected from the group consisting of from at least15 to the length of the nucleic acid sequence from which thepolynucleotide is a subsequence of. Thus, for example, polynucleotidesof the present invention are inclusive of polynucleotides comprising atleast 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides inlength from the polynucleotides of (A)-(F). Optionally, the number ofsuch subsequences encoded by a polynucleotide of the instant embodimentcan be any integer selected from the group consisting of from 1 to 20,such as 2, 3, 4, or 5. The subsequences can be separated by any integerof nucleotides from 1 to the number of nucleotides in the sequence suchas at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

[0129] Subsequences can be made by in vitro synthetic, in vitrobiosynthetic, or in vivo recombinant methods. In optional embodiments,subsequences can be made by nucleic acid amplification. For example,nucleic acid primers will be constructed to selectively hybridize to asequence (or its complement) within, or co-extensive with, the codingregion.

[0130] The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived such as a poly (A) tail.Optionally, a subsequence from a polynucleotide encoding a polypeptidehaving at least one epitope in common with a prototype polypeptidesequence as provided in (a), above, may encode an epitope in common withthe prototype sequence. Alternatively, the subsequence may not encode anepitope in common with the prototype sequence but can be used to isolatethe larger sequence by, for example, nucleic acid hybridization with thesequence from which it is derived. Subsequences can be used to modulateor detect gene expression by introducing into the subsequences compoundswhich bind, intercalate, cleave and/or crosslink to nucleic acids.Exemplary compounds include acridine, psoralen, phenanthroline,naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.

[0131] H. Polynucleotides From a Full-length Enriched cDNA LibraryHaving the Physico-Chemical Property of Selectively Hybridizing to aPolynucleotide of (A)-(G)

[0132] As indicated in (h), above, the present invention provides anisolated polynucleotide from a full-length enriched cDNA library havingthe physico-chemical property of selectively hybridizing to apolynucleotide of paragraphs (A), (B), (C), (D), (E), (F), or (G) asdiscussed above. Methods of constructing full-length enriched cDNAlibraries are known in the art and discussed briefly below. The cDNAlibrary comprises at least 50% to 95% full-length sequences (forexample, at least 50%, 60%, 70%, 80%, 90%, or 95% full-lengthsequences). The cDNA library can be constructed from a variety oftissues from a monocot or dicot at a variety of developmental stages.Exemplary species include maize, wheat, rice, canola, soybean, cotton,sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley, and rice.Methods of selectively hybridizing, under selective hybridizationconditions, a polynucleotide from a full-length enriched library to apolynucleotide of the present invention are known to those of ordinaryskill in the art. Any number of stringency conditions can be employed toallow for selective hybridization. In optional embodiments, thestringency allows for selective hybridization of sequences having atleast 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity over thelength of the hybridized region. Full-length enriched cDNA libraries canbe normalized to increase the representation of rare sequences.

[0133] I. Polynucleotide Products Made by a cDNA Isolation Process

[0134] As indicated in (I), above, the present invention provides anisolated polynucleotide made by the process of: 1) providing afull-length enriched nucleic acid library, 2) selectively hybridizingthe polynucleotide to a polynucleotide of paragraphs (A), (B), (C), (D),(E), (F), (G, or (H) as discussed above, and thereby isolating thepolynucleotide from the nucleic acid library. Full-length enrichednucleic acid libraries are constructed as discussed in paragraph (G) andbelow. Selective hybridization conditions are as discussed in paragraph(G). Nucleic acid purification procedures are well known in the art.Purification can be conveniently accomplished using solid-phase methods;such methods are well known to those of skill in the art and kits areavailable from commercial suppliers such as Advanced Biotechnologies(Surrey, UK). For example, a polynucleotide of paragraphs (A)-(H) can beimmobilized to a solid support such as a membrane, bead, or particle.See, e.g., U.S. Pat. No. 5,667,976. The polynucleotide product of thepresent process is selectively hybridized to an immobilizedpolynucleotide and the solid support is subsequently isolated fromnon-hybridized polynucleotides by methods including, but not limited to,centrifugation, magnetic separation, filtration, electrophoresis, andthe like.

[0135] Construction of Nucleic Acids

[0136] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot such as corn, rice, or wheat, or a dicot such as soybean.

[0137] The nucleic acids may conveniently comprise sequences in additionto a polynucleotide of the present invention. For example, amulti-cloning site comprising one or more endonuclease restriction sitesmay be inserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. A polynucleotideof the present invention can be attached to a vector, adapter, or linkerfor cloning and/or expression of a polynucleotide of the presentinvention. Additional sequences may be added to such cloning and/orexpression sequences to optimize their function in cloning and/orexpression, to aid in isolation of the polynucleotide, or to improve theintroduction of the polynucleotide into a cell. Typically, the length ofa nucleic acid of the present invention less the length of itspolynucleotide of the present invention is less than 20 kilobase pairs,often less than 15 kb, and frequently less than 10 kb. Use of cloningvectors, expression vectors, adapters, and linkers is well known andextensively described in the art. For a description of various nucleicacids see, for example, Stratagene Cloning Systems, Catalogs 1999 (LaJolla, Calif.); and, Amersham Life Sciences, Inc, Catalog '99 (ArlingtonHeights, Ill.).

[0138] A. Recombinant Methods for Constructing Nucleic Acids

[0139] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. Isolation of RNA, andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. See, e.g., Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and,Current Protocols in Molecular Biology, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

[0140] A1. Full-length Enriched cDNA Libraries

[0141] A number of cDNA synthesis protocols have been described whichprovide enriched full-length cDNA libraries. Enriched full-length cDNAlibraries are constructed to comprise at least 60%, and more preferablyat least 70%, 80%, 90% or 95% full-length inserts amongst clonescontaining inserts. The length of insert in such libraries can be atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or more kilobase pairs. Vectors toaccommodate inserts of these sizes are known in the art and availablecommercially. See, e.g., Stratagene's lambda ZAP Express (cDNA cloningvector with 0 to 12 kb cloning capacity). An exemplary method ofconstructing a greater than 95% pure full-length cDNA library isdescribed by Carninci et al., Genomics, 37:327-336 (1996). Other methodsfor producing full-length libraries are known in the art. See, e.g.,Edery et al., Mol. Cell Biol.,15 (6):3363-3371 (1995); and, PCTApplication WO 96/34981.

[0142] A2. Normalized or Subtracted cDNA Libraries

[0143] A non-normalized cDNA library represents the mRNA population ofthe tissue it was made from. Since unique clones are out-numbered byclones derived from highly expressed genes their isolation can belaborious. Normalization of a cDNA library is the process of creating alibrary in which each clone is more equally represented. Construction ofnormalized libraries is described in Ko, Nucl. Acids. Res., 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.,88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685, 5,482,845, and 5,637,685.In an exemplary method described by Soares et al., normalizationresulted in reduction of the abundance of clones from a range of fourorders of magnitude to a narrow range of only 1 order of magnitude.Proc. Natl. Acad. Sci. USA, 91:9228-9232 (1994).

[0144] Subtracted cDNA libraries are another means to increase theproportion of less abundant cDNA species. In this procedure, cDNAprepared from one pool of mRNA is depleted of sequences present in asecond pool of mRNA by hybridization. The cDNA:mRNA hybrids are removedand the remaining un-hybridized cDNA pool is enriched for sequencesunique to that pool. See, Foote et al. in, Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho andZarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. AcidsRes., 16(22):10937 (1988); Current Protocols in Molecular Biology,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995); and, Swaroop et al., Nucl. Acids Res., 19)8):1954 (1991).cDNA subtraction kits are commercially available. See, e.g., PCR-Select(Clontech, Palo Alto, Calif.).

[0145] To construct genomic libraries, large segments of genomic DNA aregenerated by fragmentation, e.g. using restriction endonucleases, andare ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Vols. 1-3 (1989), Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

[0146] The cDNA or genomic library can be screened using a probe basedupon the sequence of a polynucleotide of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent.

[0147] The nucleic acids of interest can also be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to make nucleic acids to use asprobes for detecting the presence of the desired mRNA in samples, fornucleic acid sequencing, or for other purposes. The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

[0148] PCR-based screening methods have been described. Wilfinger et al.describe a PCR-based method in which the longest cDNA is identified inthe first step so that incomplete clones can be eliminated from study.BioTechniques, 22(3): 481-486 (1997). Such methods are particularlyeffective in combination with a full-length cDNA constructionmethodology, above.

[0149] B. Synthetic Methods for Constructing Nucleic Acids

[0150] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99(1979); the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et al.,Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramiditetriester method described by Beaucage and Caruthers, Tetra. Letts.22(20): 1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984); and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis generally produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill willrecognize that while chemical synthesis of DNA is best employed forsequences of about 100 bases or less, longer sequences may be obtainedby the ligation of shorter sequences.

Recombinant Expression Cassettes

[0151] The present invention further provides recombinant expressioncassettes comprising a nucleic acid of the present invention. A nucleicacid sequence coding for the desired polypeptide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength polypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

[0152] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0153] A plant promoter fragment can be employed which will directexpression of a polynucleotide of the present invention in all tissuesof a regenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′- promoter derivedfrom T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, theSmas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat.No. 5,683,439), the Nos promoter, the pEmu promoter, the rubiscopromoter, and the GRP1-8 promoter.

[0154] Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

[0155] Examples of promoters under developmental control includepromoters that initiate transcription only, or preferentially, incertain tissues, such as leaves, roots, fruit, seeds, or flowers.Exemplary promoters include the anther specific promoter 5126 (U.S. Pat.Nos. 5,689,049 and 5,689,051), glob-1 promoter, and gamma-zein promoter.The operation of a promoter may also vary depending on its location inthe genome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

[0156] Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inrecombinant expression cassettes to drive expression of antisensenucleic acids to reduce, increase, or alter concentration and/orcomposition of the proteins of the present invention in a desiredtissue. Thus, in some embodiments, the nucleic acid construct willcomprise a promoter, functional in a plant cell, operably linked to apolynucleotide of the present invention. Promoters useful in theseembodiments include the endogenous promoters driving expression of apolypeptide of the present invention.

[0157] In some embodiments, isolated nucleic acids which serve aspromoter or enhancer elements can be introduced in the appropriateposition (generally upstream) of a non-heterologous form of apolynucleotide of the present invention so as to up or down regulateexpression of a polynucleotide of the present invention. For example,endogenous promoters can be altered in vivo by mutation, deletion,and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling etal., WO 93/22443), or isolated promoters can be introduced into a plantcell in the proper orientation and distance from a cognate gene of apolynucleotide of the present invention so as to control the expressionof the gene. Gene expression can be modulated under conditions suitablefor plant growth so as to alter the total concentration and/or alter thecomposition of the polypeptides of the present invention in plant cell.Thus, the present invention provides compositions, and methods formaking, heterologous promoters and/or enhancers operably linked to anative, endogenous (i.e., non-heterologous) form of a polynucleotide ofthe present invention.

[0158] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0159] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,Mol. Cell Biol. 8: 4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. See generally, The Maize Handbook, Chapter116, Freeling and Walbot, Eds., Springer, New York (1994). The vectorcomprising the sequences from a polynucleotide of the present inventionwill typically comprise a marker gene which confers a selectablephenotype on plant cells. Typical vectors useful for expression of genesin higher plants are well known in the art and include vectors derivedfrom the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciensdescribed by Rogers et al., Meth. in Enzymol., 153:253-277 (1987).

[0160] A polynucleotide of the present invention can be expressed ineither sense or anti-sense orientation as desired. It will beappreciated that control of gene expression in either sense oranti-sense orientation can have a direct impact on the observable plantcharacteristics. Antisense technology can be conveniently used toinhibit gene expression in plants. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed. Theconstruct is then transformed into plants and the antisense strand ofRNA is produced. In plant cells, it has been shown that antisense RNAinhibits gene expression by preventing the accumulation of mRNA whichencodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Nat'l.Acad. Sci. (USA) 85: 8805-8809 (1988); and Shewmaker, Hiatt, et al.,U.S. Pat. No. 5,759,829.

[0161] Another method of suppression is sense suppression (i.e.,co-supression). Introduction of nucleic acid configured in the senseorientation has been shown to be an effective means by which to blockthe transcription of target genes. For an example of the use of thismethod to modulate expression of endogenous genes see, Napoli et al.,The Plant Cell 2: 279-289 (1990) and U.S. Pat. No. 5,034,323.

[0162] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334: 585-591 (1988).

[0163] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J Am Chem Soc (1987) 109:1241-1243). Meyer, R. B.,et al., J Am Chem Soc (1989) 111:8517-8519, effect covalent crosslinkingto a target nucleotide using an alkylating agent complementary to thesingle-stranded target nucleotide sequence. A photoactivatedcrosslinking to single-stranded oligonucleotides mediated by psoralenwas disclosed by Lee, B. L., et al., Biochemistry (1988) 27:3197-3203.Use of crosslinking in triple-helix forming probes was also disclosed byHome, et al., J Am Chem Soc (1990) 112:2435-2437. Use of N4,N4-ethanocytosine as an alkylating agent to crosslink to single-strandedoligonucleotides has also been described by Webb and Matteucci, J AmChem Soc (1986) 108:2764-2765; Nucleic Acids Res (1986) 14:7661-7674;Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991). Various compounds tobind, detect, label, and/or cleave nucleic acids are known in the art.See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908;5,256,648; and, 5,681,941.

Proteins

[0164] The isolated proteins of the present invention comprise apolypeptide having at least 10 amino acids from a polypeptide of thepresent invention (or conservative variants thereof) such as thoseencoded by any one of the polynucleotides of the present invention asdiscussed more fully above. The proteins of the present invention orvariants thereof can comprise any number of contiguous amino acidresidues from a polypeptide of the present invention, wherein thatnumber is selected from the group of integers consisting of from 10 tothe number of residues in a full-length polypeptide of the presentinvention. Optionally, this subsequence of contiguous amino acids is atleast 15, 20, 25, 30, 35, or 40 amino acids in length, often at least50, 60, 70, 80, or 90 amino acids in length. Further, the number of suchsubsequences can be any integer selected from the group consisting offrom 1 to 20, such as 2, 3, 4, or 5.

[0165] The present invention further provides a protein comprising apolypeptide having a specified sequence identity/similarity with apolypeptide of the present invention. The percentage of sequenceidentity/similarity is an integer selected from the group consisting offrom 50 to 99. Exemplary sequence identity/similarity values include55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%. Sequence identity canbe determined using, for example, the GAP, CLUSTALW, or BLASTalgorithms.

[0166] As those of skill will appreciate, the present inventionincludes, but is not limited to, catalytically active polypeptides ofthe present invention (i.e., enzymes). Catalytically active polypeptideshave a specific activity of at least 20%, 30%, or 40%, and preferably atleast 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%that of the native (non-synthetic), endogenous polypeptide. Further, thesubstrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40%, or 50%, that of thenative (non-synthetic), endogenous polypeptide; and more preferably atleast 60%, 70%, 80%, or 90%. Methods of assaying and quantifyingmeasures of enzymatic activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art. Generally,the proteins of the present invention will, when presented as animmunogen, elicit production of an antibody specifically reactive to apolypeptide of the present invention. Further, the proteins of thepresent invention will not bind to antisera raised against a polypeptideof the present invention which has been fully immunosorbed with the samepolypeptide. Immunoassays for determining binding are well known tothose of skill in the art. A preferred immunoassay is a competitiveimmunoassay. Thus, the proteins of the present invention can be employedas immunogens for constructing antibodies immunoreactive to a protein ofthe present invention for such exemplary utilities as immunoassays orprotein purification techniques.

Expression of Proteins in Host Cells

[0167] Using the nucleic acids of the present invention, one may expressa protein of the present invention in a recombinantly engineered cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The cells produce the protein in a non-natural condition (e.g., inquantity, composition, location, and/or time), because they have beengenetically altered through human intervention to do so.

[0168] It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of a nucleicacid encoding a protein of the present invention. No attempt to describein detail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

[0169] In brief summary, the expression of isolated nucleic acidsencoding a protein of the present invention will typically be achievedby operably linking, for example, the DNA or cDNA to a promoter (whichis either constitutive or regulatable), followed by incorporation intoan expression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high-levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located purification sequences. Restriction sites ortermination codons can also be introduced.

Synthesis of Proteins

[0170] The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewartet al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greater lengthmay be synthesized by condensation of the amino and carboxy termini ofshorter fragments. Methods of forming peptide bonds by activation of acarboxy terminal end (e.g., by the use of the coupling reagentN,N′-dicycylohexylcarbodiimide) are known to those of skill.

Purification of Proteins

[0171] The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

[0172] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. The protein may then be isolatedfrom cells expressing the protein and further purified by standardprotein chemistry techniques as described herein. Detection of theexpressed protein is achieved by methods known in the art and include,for example, radioimmunoassays, Western blotting techniques orimmunoprecipitation.

Introduction of Nucleic Acids Into Host Cells

[0173] The method of introducing a nucleic acid of the present inventioninto a host cell is not critical to the instant invention.Transformation or transfection methods are conveniently used.Accordingly, a wide variety of methods have been developed to insert aDNA sequence into the genome of a host cell to obtain the transcriptionand/or translation of the sequence to effect phenotypic changes in theorganism. Thus, any method which provides for effective introduction ofa nucleic acid may be employed.

[0174] A. Plant Transformation

[0175] A nucleic acid comprising a polynucleotide of the presentinvention is optionally introduced into a plant. Generally, thepolynucleotide will first be incorporated into a recombinant expressioncassette or vector. Isolated nucleic acid acids of the present inventioncan be introduced into plants according to techniques known in the art.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical, scientific, and patentliterature. See, for example, Weising et al., Ann. Rev. Genet 22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, polyethylene glycol (PEG), poration, particlebombardment, silicon fiber delivery, or microinjection of plant cellprotoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNATransfer into Intact Plant Cells Via Microprojectile Bombardment.pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods.eds. O. L. Gamborg and G. C. Phillips. Springer-Verlag Berlin HeidelbergNew York, 1995; see, U.S. Pat. No. 5,990,387. The introduction of DNAconstructs using PEG precipitation is described in Paszkowski et al.,Embo J. 3: 2717-2722 (1984). Electroporation techniques are described inFromm et al., Proc. Natl. Acad. Sci. (USA) 82: 5824 (1985). Ballistictransformation techniques are described in Klein et al., Nature 327:70-73 (1987).

[0176]Agrobacterium tumefaciens-mediated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233: 496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci.(USA) 80: 4803 (1983); and, Plant Molecular Biology: A LaboratoryManual, Chapter 8, Clark, Ed., Springer-Verlag, Berlin (1997). The DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria. See, U.S. Pat. No.5,591,616. Although Agrobacterium is useful primarily in dicots, certainmonocots can be transformed by Agrobacterium. For instance,Agrobacterium transformation of maize is described in U.S. Pat. No.5,550,318.

[0177] Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J,.In: DNA Cloning, Vol. 11, D. M. Glover, Ed., Oxford, IRI Press, 1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25: 1353 (1984)),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci.,(USA) 87:1228 (1990).

[0178] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codinggenes can be obtained by injection of the DNA into reproductive organsof a plant as described by Pena et al., Nature, 325.:274 (1987). DNA canalso be injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plantviruses that can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

[0179] B. Transfection of Prokaryotes, Lower Eukaryotes, and AnimalCells

[0180] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

Transgenic Plant Regeneration

[0181] Plant cells which directly result or are derived from the nucleicacid introduction techniques can be cultured to regenerate a whole plantwhich possesses the introduced genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium. Plants cells can be regenerated, e.g., from single cells,callus tissue or leaf discs according to standard plant tissue culturetechniques. It is well known in the art that various cells, tissues, andorgans from almost any plant can be successfully cultured to regeneratean entire plant. Plant regeneration from cultured protoplasts isdescribed in Evans et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, Macmillan Publishing Company, New York,pp.124-176 (1983); and Binding, Regeneration of Plants, PlantProtoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).

[0182] The regeneration of plants from either single plant protoplastsor various explants is well known in the art. See, for example, Methodsfor Plant Molecular Biology, A. Weissbach and H. Weissbach, eds.,Academic Press, Inc., San Diego, Calif. (1988). This regeneration andgrowth process includes the steps of selection of transformant cells andshoots, rooting the transformant shoots and growth of the plantlets insoil. For maize cell culture and regeneration see generally, The MaizeHandbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn andCorn Improvement, 3^(rd) edition, Sprague and Dudley Eds., AmericanSociety of Agronomy, Madison, Wis. (1988). For transformation andregeneration of maize see, Gordon-Kamm et al., The Plant Cell, 2:603-618(1990).

[0183] The regeneration of plants containing the polynucleotide of thepresent invention and introduced by Agrobacterium from leaf explants canbe achieved as described by Horsch et al., Science, 227:1229-1231(1985). In this procedure, transformants are grown in the presence of aselection agent and in a medium that induces the regeneration of shootsin the plant species being transformed as described by Fraley et al.,Proc. Natl. Acad. Sci. (U.S.A.), 80:4803 (1983). This proceduretypically produces shoots within two to four weeks and thesetransformant shoots are then transferred to an appropriate root-inducingmedium containing the selective agent and an antibiotic to preventbacterial growth. Transgenic plants of the present invention may befertile or sterile.

[0184] One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences. Transgenic plants expressing a polynucleotide ofthe present invention can be screened for transmission of the nucleicacid of the present invention by, for example, standard immunoblot andDNA detection techniques. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0185] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated.

Modulating Polypeptide Levels and/or Composition

[0186] The present invention further provides a method for modulating oraltering (i.e., increasing or decreasing) the concentration and/or ratioof the polypeptides of the present invention in a plant or part thereof.The method comprises introducing into a plant cell a recombinantexpression cassette comprising a polynucleotide of the present inventionas described above to obtain a transgenic plant cell, culturing thetransgenic plant cell under transgenic plant cell growing conditions,and inducing or repressing expression of a polynucleotide of the presentinvention in the transgenic plant for a time sufficient to modulateconcentration and/or the ratios of the polypeptides in the transgenicplant or plant part.

[0187] In some embodiments, the concentration and/or ratios ofpolypeptides of the present invention in a plant may be modulated byaltering, in vivo or in vitro, the promoter of a gene to up- ordown-regulate gene expression. In some embodiments, the coding regionsof native genes of the present invention can be altered viasubstitution, addition, insertion, or deletion to decrease activity ofthe encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarlinget al., WO 93/22443. And in some embodiments, an isolated nucleic acid(e.g., a vector) comprising a promoter sequence is transfected into aplant cell. Subsequently, a plant cell comprising the promoter operablylinked to a polynucleotide of the present invention is selected for bymeans known to those of skill in the art such as, but not limited to,Southern blot, DNA sequencing, or PCR analysis using primers specific tothe promoter and to the gene and detecting amplicons produced therefrom.A plant or plant part altered or modified by the foregoing embodimentsis grown under plant forming conditions for a time sufficient tomodulate the concentration and/or ratios of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart and discussed briefly, supra.

[0188] In general, concentration or the ratios of the polypeptides isincreased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% relative to a native control plant, plant part, or celllacking the aforementioned recombinant expression cassette. Modulationin the present invention may occur during and/or subsequent to growth ofthe plant to the desired stage of development. Modulating nucleic acidexpression temporally and/or in particular tissues can be controlled byemploying the appropriate promoter operably linked to a polynucleotideof the present invention in, for example, sense or antisense orientationas discussed in greater detail, supra. Induction of expression of apolynucleotide of the present invention can also be controlled byexogenous administration of an effective amount of inducing compound.Inducible promoters and inducing compounds which activate expressionfrom these promoters are well known in the art. In preferredembodiments, the polypeptides of the present invention are modulated inmonocots, particularly maize.

UTRs and Codon Preference

[0189] In general, translational efficiency has been found to beregulated by specific sequence elements in the 5′ non-coding oruntranslated region (5′ UTR) of the RNA. Positive sequence motifsinclude translational initiation consensus sequences (Kozak, NucleicAcids Res. 15:8125 (1987)) and the 7-methylguanosine cap structure(Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elementsinclude stable intramolecular 5′ UTR stem-loop structures (Muesing etal., Cell 48:691 (1987)) and AUG sequences or short open reading framespreceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al.,Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present inventionprovides 5′ and/or 3′ untranslated regions for modulation of translationof heterologous coding sequences.

[0190] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host such as tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50, or 100.

Sequence Shuffling

[0191] The present invention provides methods for sequence shufflingusing polynucleotides of the present invention, and compositionsresulting therefrom. Sequence shuffling is described in PCT publicationNo. WO 97/20078. See also, Zhang, J.-H., et al. Proc. Natl. Acad. Sci.USA 94:4504-4509 (1997). Generally, sequence shuffling provides a meansfor generating libraries of polynucleotides having a desiredcharacteristic which can be selected or screened for. Libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides which comprise sequence regions which havesubstantial sequence identity and can be homologously recombined invitro or in vivo. The population of sequence-recombined polynucleotidescomprises a subpopulation of polynucleotides which possess desired oradvantageous characteristics and which can be selected by a suitableselection or screening method. The characteristics can be any propertyor attribute capable of being selected for or detected in a screeningsystem, and may include properties of: an encoded protein, atranscriptional element, a sequence controlling transcription, RNAprocessing, RNA stability, chromatin conformation, translation, or otherexpression property of a gene or transgene, a replicative element, aprotein-binding element, or the like, such as any feature which confersa selectable or detectable property. In some embodiments, the selectedcharacteristic will be a decreased K_(m) and/or increased K_(cat) overthe wild-type protein as provided herein. In other embodiments, aprotein or polynucleotide generated from sequence shuffling will have aligand binding affinity greater than the non-shuffled wild-typepolynucleotide. The increase in such properties can be at least 110%,120%, 130%, 140% or at least 150% of the wild-type value.

Generic and Consensus Sequences

[0192] Polynucleotides and polypeptides of the present invention furtherinclude those having: (a) a generic sequence of at least two homologouspolynucleotides or polypeptides, respectively, of the present invention;and, (b) a consensus sequence of at least three homologouspolynucleotides or polypeptides, respectively, of the present invention.The generic sequence of the present invention comprises each species ofpolypeptide or polynucleotide embraced by the generic polypeptide orpolynucleotide sequence, respectively. The individual speciesencompassed by a polynucleotide having an amino acid or nucleic acidconsensus sequence can be used to generate antibodies or produce nucleicacid probes or primers to screen for homologs in other species, genera,families, orders, classes, phyla, or kingdoms. For example, apolynucleotide having a consensus sequence from a gene family of Zeamays can be used to generate antibody or nucleic acid probes or primersto other Gramineae species such as wheat, rice, or sorghum.Alternatively, a polynucleotide having a consensus sequence generatedfrom orthologous genes can be used to identify or isolate orthologs ofother taxa. Typically, a polynucleotide having a consensus sequence willbe at least 9, 10, 15, 20, 25, 30, or 40 amino acids in length, or 20,30, 40, 50, 100, or 150 nucleotides in length. As those of skill in theart are aware, a conservative amino acid substitution can be used foramino acids which differ amongst aligned sequence but are from the sameconservative substitution group as discussed above. Optionally, no morethan 1 or 2 conservative amino acids are substituted for each 10 aminoacid length of consensus sequence.

[0193] Similar sequences used for generation of a consensus or genericsequence include any number and combination of allelic variants of thesame gene, orthologous, or paralogous sequences as provided herein.Optionally, similar sequences used in generating a consensus or genericsequence are identified using the BLAST algorithm's smallest sumprobability (P(N)). Various suppliers of sequence-analysis software arelisted in chapter 7 of Current Protocols in Molecular Biology, F. M.Ausubel et al., Eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30).A polynucleotide sequence is considered similar to a reference sequenceif the smallest sum probability in a comparison of the test nucleic acidto the reference nucleic acid is less than about 0.1, more preferablyless than about 0.01, or 0.001, and most preferably less than about0.0001, or 0.00001. Similar polynucleotides can be aligned and aconsensus or generic sequence generated using multiple sequencealignment software available from a number of commercial suppliers suchas the Genetics Computer Group's (Madison, Wis.) PILEUP software, VectorNTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.)SEQUENCHER. Conveniently, default parameters of such software can beused to generate consensus or generic sequences.

Machine Applications

[0194] The present invention provides processes for modeling oranalyzing the polynucleotides and polypeptides of the present invention.

[0195] The present invention provides a process of identifying acandidate homologue (i.e., an ortholog or paralog) of a polynucleotideor polypeptide of the present invention. The process comprises enteringsequence data of a polynucleotide or polypeptide of the presentinvention into a machine having a hardware or software sequence analysissystem, developing data structures to facilitate access to the sequencedata, manipulating the data to analyze the structure the polynucleotideor polypeptide, and displaying the results of the analysis. A candidatehomologue has a statistically significant probability of having the samebiological function (e.g., catalyzes the same reaction, binds tohomologous proteins/nucleic acids, has a similar structural role) as thereference sequence to which it is compared. Accordingly, thepolynucleotides and polypeptides of the present invention have utilityin identifying homologs in animals or other plant species, particularlythose in the family Gramineae such as, but not limited to, sorghum,wheat, or rice.

[0196] The process of the present invention comprises obtaining datarepresenting a polynucleotide or polypeptide test sequence. Testsequences can be obtained from a nucleic acid of an animal or plant.Test sequences can be obtained directly or indirectly from sequencedatabases including, but not limited to, those such as: GenBank, EMBL,GenSeq, SWISS-PROT, or those available on-line via the UK Human GenomeMapping Project (HGMP) GenomeWeb. In some embodiments the test sequenceis obtained from a plant species other than maize whose function isuncertain but will be compared to the test sequence to determinesequence similarity or sequence identity. The test sequence data isentered into a machine, such as a computer, containing: i) datarepresenting a reference sequence and, ii) a hardware or softwaresequence comparison system to compare the reference and test sequencefor sequence similarity or identity.

[0197] Exemplary sequence comparison systems are provided for insequence analysis software such as those provided by the GeneticsComputer Group (Madison, Wis.) or InforMax (Bethesda, Md.), orIntelligenetics (Mountain View, Calif.). Optionally, sequence comparisonis established using the BLAST or GAP suite of programs. Generally, asmallest sum probability value (P(N)) of less than 0.1, oralternatively, less than 0.01, 0.001, 0.0001, or 0.00001 using the BLAST2.0 suite of algorithms under default parameters identifies the testsequence as a candidate homologue (i.e., an allele, ortholog, orparalog) of the reference sequence. Those of skill in the art willrecognize that a candidate homologue has an increased statisticalprobability of having the same or similar function as the gene/proteinrepresented by the test sequence.

[0198] The reference sequence can be the sequence of a polypeptide or apolynucleotide of the present invention. The reference or test sequenceis each optionally at least 25 amino acids or at least 100 nucleotidesin length. The length of the reference or test sequences can be thelength of the polynucleotide or polypeptide described, respectively,above in the sections entitled “Nucleic Acids” (particularly section(g)), and “Proteins”. As those of skill in the art are aware, thegreater the sequence identity/similarity between a reference sequence ofknown function and a test sequence, the greater the probability that thetest sequence will have the same or similar function as the referencesequence. The results of the comparison between the test and referencesequences are outputted (e.g., displayed, printed, recorded) via any oneof a number of output devices and/or media (e.g., computer monitor, hardcopy, or computer readable medium).

Detection of Nucleic Acids

[0199] The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of containing a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of maize. In someembodiments, a cognate gene of a polynucleotide of the present inventionor portion thereof can be amplified prior to the step of contacting thenucleic acid sample with a polynucleotide of the present invention. Thenucleic acid sample is contacted with the polynucleotide to form ahybridization complex. The polynucleotide hybridizes under stringentconditions to a gene encoding a polypeptide of the present invention.Formation of the hybridization complex is used to detect a gene encodinga polypeptide of the present invention in the nucleic acid sample. Thoseof skill will appreciate that an isolated nucleic acid comprising apolynucleotide of the present invention should lack cross-hybridizingsequences in common with non-target genes that would yield a falsepositive result. Detection of the hybridization complex can be achievedusing any number of well-known methods. For example, the nucleic acidsample, or a portion thereof, may be assayed by hybridization formatsincluding but not limited to, solution phase, solid phase, mixed phase,or in situ hybridization assays.

[0200] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads,fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Otherlabels include ligands which bind to antibodies labeled withfluorophores, chemiluminescent agents, and enzymes. Labeling the nucleicacids of the present invention is readily achieved such as by the use oflabeled PCR primers.

[0201] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

EXAMPLE 1

[0202] This example describes the construction of a cDNA library.

[0203] Total RNA can be isolated from maize tissues with TRIzol Reagent(Life Technology Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Chomczynski, P., and Sacchi, N. Anal. Biochem. 162, 156(1987)). In brief, plant tissue samples are pulverized in liquidnitrogen before the addition of the TRizol Reagent, and then furtherhomogenized with a mortar and pestle. Addition of chloroform followed bycentrifugation is conducted for separation of an aqueous phase and anorganic phase. The total RNA is recovered by precipitation withisopropyl alcohol from the aqueous phase.

[0204] The selection of poly(A)+ RNA from total RNA can be performedusing PolyATtract® system (Promega Corporation. Madison, Wis.).Biotinylated oligo(dT) primers are used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids are captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA is thenwashed at high stringency conditions and eluted by RNase-free deionizedwater.

[0205] cDNA synthesis and construction of unidirectional cDNA librariescan be accomplished using the SuperScript Plasmid System (LifeTechnology Inc. Gaithersburg, Md.). The first strand of cDNA issynthesized by priming an oligo(dT) primer containing a Not I site. Thereaction is catalyzed by SuperScript Reverse Transcriptase II at 45° C.The second strand of cDNA is labeled with alpha-³²P-dCTP and a portionof the reaction analyzed by agarose gel electrophoresis to determinecDNA sizes. cDNA molecules smaller than 500 base pairs and unligatedadapters are removed by Sephacryl-S400 chromatography. The selected cDNAmolecules are ligated into pSPORT1 vector in between of Not I and Sal Isites.

[0206] Alternatively, cDNA libraries can be prepared by any one of manymethods available. For example, the cDNAs may be introduced into plasmidvectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectorsaccording to the manufacturer's protocol (Stratagene Cloning Systems, LaJolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmidlibraries according to the protocol provided by Stratagene. Uponconversion, cDNA inserts will be contained in the plasmid vectorpBluescript. In addition, the cDNAs may be introduced directly intoprecut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (NewEngland Biolabs), followed by transfection into DH10B cells according tothe manufacturer's protocol (GIBCO BRL Products). Once the cDNA insertsare in plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert cDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

EXAMPLE 2

[0207] This method describes construction of a full-length enriched cDNAlibrary.

[0208] An enriched full-length cDNA library can be constructed using oneof two variations of the method of Carninci et al. Genomics 37: 327-336,1996. These variations are based on chemical introduction of a biotingroup into the diol residue of the 5′ cap structure of eukaryotic mRNAto select full-length first strand cDNA. The selection occurs bytrapping the biotin residue at the cap sites using streptavidin-coatedmagnetic beads followed by RNase I treatment to eliminate incompletelysynthesized cDNAs. Second strand cDNA is synthesized using establishedprocedures such as those provided in Life Technologies' (Rockville, Md.)“SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning” kit.Libraries made by this method have been shown to contain 50% to 70%full-length cDNAs.

[0209] The first strand synthesis methods are detailed below. Anasterisk denotes that the reagent was obtained from Life Technologies,Inc. A. First strand cDNA synthesis method 1 (with trehalose) mRNA (10ug) 25 μl *Not I primer (5 ug) 10 μl *5 × 1^(st) strand buffer 43 μl*0.1 m DTT 20 μl *dNTP mix 10 mm 10 μl BSA 10 ug/μl  1 μl Trehalose(saturated) 59.2 μl   RNase inhibitor (Promega) 1.8 μl  *Superscript IIRT 200 u/μl 20 μl 100% glycerol 18 μl Water  7 μl

[0210] The mRNA and Not I primer are mixed and denatured at 65° C. for10 min. They are then chilled on ice and other components added to thetube. Incubation is at 45° C. for 2 min. Twenty microliters of RT(reverse transcriptase) is added to the reaction and start program onthe thermocycler (MJ Research, Waltham, Mass.): Step 1 45° C. 10 minStep 2 45° C. −0.3° C./cycle, 2 seconds/cycle Step 3 go to 2 for 33cycles Step 4 35° C. 5 min Step 5 45° C. 5 min Step 6 45° C. 0.2°C./cycle, 1 sec/cycle Step 7 go to 7 for 49 cycles Step 8 55° C. 0.1°C./cycle, 12 sec/cycle Step 9 go to 8 for 49 cycles Step 10 55° C. 2 minStep 11 60° C. 2 min Step 12 go to 11 for 9 times Step 13 4° C. foreverStep 14 end

[0211] B. First strand cDNA synthesis method 2 mRNA (10 μg) 25 μl water30 μl *Not I adapter primer (5 μg) 10 μl 65° C. for 10 min, chill onice, then add following reagents, *5x first buffer 20 μl *0.1M DTT 10 μl*10 mM dNTP mix  5 μl

[0212] Incubate at 45° C. for 2 min, then add 10 μl of *Superscript IIRT (200 u/μl), start the following program: Step 1 45° C. for 6 sec,−0.1° C./cycle Step 2 go to 1 for 99 additional cycles Step 3 35° C. for5 min Step 4 45° C. for 60 min Step 5 50° C. for 10 min Step 6 4° C.forever Step 7 end

[0213] After the 1^(st) strand cDNA synthesis, the DNA is extracted byphenol according to standard procedures, and then precipitated in NaOAcand ethanol, and stored in −20° C.

[0214] C. Oxidization of the diol group of mRNA for biotin labeling

[0215] First strand cDNA is spun down and washed once with 70% EtOH. Thepellet resuspended in 23.2 μl of DEPC treated water and put on ice.Prepare 100 mM of NalO4 freshly, and then add the following reagents:mRNA: 1^(st) cDNA (start with 20 μg mRNA) 46.4 μl  100 mM NalO4 (freshlymade) 2.5 μl NaOAc 3M pH 4.5 1.1 μl

[0216] To make 100 mM NalO4, use 21.39 μg of NaIO4 for 1 μl of water.Wrap the tube in a foil and incubate on ice for 45 min. After theincubation, the reaction is then precipitated in: 5M NaCl 10 μl 20% SDS0.5 μl  isopropanol 61 μl

[0217] Incubate on ice for at least 30 min, then spin it down at maxspeed at 4° C. for 30 min and wash once with 70% ethanol and then 80%EtOH.

[0218] D. Biotinylation of the mRNA diol group

[0219] Resuspend the DNA in 110 μl DEPC treated water, then add thefollowing reagents: 20% SDS 5 μl 2M NaOAc pH 6.1 5 μl 10 mm biotinhydrazide (freshly made) 300 μl 

[0220] Wrap in a foil and incubate at room temperature overnight.

[0221] E. RNase I treatment

[0222] Precipitate DNA in: 5M NaCl 10 μl 2M NaOAc pH 6.1 75 μlbiotinylated mRNA:cDNA 420 μl  100% EtOH (2.5 Vol) 1262.5 μl   

[0223] (Perform this precipitation in two tubes and split the 420 μl ofDNA into 210 μl each, add 5 μl of 5 M NaCl, 37.5 μl of 2M NaOAc pH 6.1,and 631.25 μl of 100% EtOH). Store at −20° C. for at least 30 min. Spinthe DNA down at 4° C. at maximal speed for 30 min. and wash with 80%EtOH twice, then dissolve DNA in 70 μl RNase free water. Pool two tubesand end up with 140 μl. Add the following reagents: RNase One 10 U/μl 40μl 1^(st) cDNA:RNA 140 μl  10X buffer 20 μl

[0224] Incubate at 37° C. for 15 min. Add 5 μl of 40 μg/μl yeast tRNA toeach sample for capturing.

[0225] F. Full length 1^(st) cDNA capturing

[0226] Blocking the beads with yeast tRNA: Beads 1 ml Yeast tRNA 40μg/μl 5 μl

[0227] Incubate on ice for 30 min with mixing, wash 3 times with 1 ml of2M NaCl, 50 mm EDTA, pH 8.0.

[0228] Resuspend the beads in 800 μl of 2M NaCl, 50 mm EDTA, pH 8.0, addRNase I treated sample 200 μl, and incubate the reaction for 30 min atroom temperature. Capture the beads using the magnetic stand, save thesupernatant, and start following washes:

[0229] 2 washes with 2M NaCl, 50 mm EDTA, pH 8.0, 1 ml each time,

[0230] 1 wash with 0.4% SDS, 50 μg/ml tRNA,

[0231] 1 wash with 10 mm Tris-Cl pH 7.5, 0.2 mm EDTA, 10 mm NaCl, 20%glycerol,

[0232] 1 wash with 50 μg/ml tRNA,

[0233] 1 wash with 1^(st) cDNA buffer

[0234] G. Second strand cDNA synthesis

[0235] Resuspend the beads in: *5X first buffer 8 μl *0.1 mM DTT 4 μl*10 mm dNTP mix 8 μl *5X 2nd buffer 60 μl *E. coli Ligase 10 U/μl 2 μl*E. coli DNA polymerase 10 U/μl 8 μl *E. coli RNaseH 2 U/μl 2 μl P32dCTP 10 μci/μl 2 μl Or water up to 300 μl 208 μl

[0236] Incubate at 16° C. for 2 hr with mixing the reaction in every 30min. Add 4 μl of T4 DNA polymerase and incubate for additional 5 min at16° C.

[0237] Elute ₂nd cDNA from the beads. Use a magnetic stand to separatethe 2^(nd) cDNA from the beads, then resuspend the beads in 200 μl ofwater, and then separate again, pool the samples (about 500 μl), Add 200μl of water to the beads, then 200 μl of phenol:chloroform, vortex, andspin to separate the sample with phenol. Pool the DNA together (about700 μl) and use phenol to clean the DNA again, DNA is then precipitatedin 2 μg of glycogen and 0.5 vol of 7.5M NH4OAc and 2 vol of 100% EtOH.Precipitate overnight. Spin down the pellet and wash with 70% EtOH,air-dry the pellet. DNA 250 μl DNA 200 μl 7.5 M NH4OAc 125 μl 7.5 MNH4OAc 100 μl 100% EtOH 750 μl 100% EtOH 600 μl glycogen 1 μg/μl 2 μlglycogen 1 μg/μl 2 μl

[0238] H. Sal I adapter ligation

[0239] Resuspend the pellet in 26 μl of water and use 1 μl for TAE gel.Set up reaction as following: 2^(nd) strand cDNA 25 μl *5X T4 DNA ligasebuffer 10 μl *Sal I adapters 10 μl *T4 DNA ligase 5 μl

[0240] Mix gently, incubate the reaction at 16° C. overnight. Add 2 μlof ligase second day and incubate at room temperature for 2 hrs(optional). Add 50 μl water to the reaction and use 100 μl of phenol toclean the DNA, 90 μl of the upper phase is transferred into a new tubeand precipitate in: Glycogen 1 μg/μl 2 μl Upper phase DNA 90 μl 7.5 MNH4OAc 50 μl 100% EtOH 300 μl

[0241] precipitate at −20° C. overnight Spin down the pellet at 4° C.and wash in 70% EtOH, dry the pellet.

[0242] I. Not I digestion 2^(nd) cDNA 41 μl *Reaction 3 buffer 5 μl *NotI 15 u/μl 4 μl

[0243] Mix gently and incubate the reaction at 37° C. for 2 hr. Add 50μl of water and 100 μl of phenol, vortex, and take 90 μl of the upperphase to a new tube, then add 50 μl of NH4OAc and 300 μl of EtOH.Precipitate overnight at −20° C.

[0244] Cloning, ligation, and transformation are performed per theSuperScript cDNA synthesis kit. (Life Technology Inc. Gaithersburg, Md.)

EXAMPLE 3

[0245] This example describes cDNA sequencing and library subtraction.

[0246] Individual colonies can be picked and DNA prepared either by PCRwith M13 forward primers and M13 reverse primers, or by plasmidisolation. cDNA clones can be sequenced using M13 reverse primers.

[0247] cDNA libraries are plated out on 22×22 cm² agar plate at densityof about 3,000 colonies per plate. The plates are incubated in a 37° C.incubator for 12-24 hours. Colonies are picked into 384-well plates by arobot colony picker, Q-bot (GENETIX Limited). These plates are incubatedovernight at 37° C. Once sufficient colonies are picked, they are pinnedonto 22×22 cm² nylon membranes using Q-bot. Each membrane holds 9,216 or36,864 colonies. These membranes are placed onto an agar plate with anappropriate antibiotic. The plates are incubated at 37° C. overnight.

[0248] After colonies are recovered on the second day, these filters areplaced on filter paper prewetted with denaturing solution for fourminutes, then incubated on top of a boiling water bath for an additionalfour minutes. The filters are then placed on filter paper prewetted withneutralizing solution for four minutes. After excess solution is removedby placing the filters on dry filter papers for one minute, the colonyside of the filters is placed into Proteinase K solution, incubated at37° C. for 40-50 minutes. The filters are placed on dry filter papers todry overnight. DNA is then cross-linked to nylon membrane by UV lighttreatment.

[0249] Colony hybridization is conducted as described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratoryManual, 2^(nd) Edition). The following probes can be used in colonyhybridization:

[0250] 1. First strand cDNA from the same tissue as the library was madefrom to remove the most redundant clones.

[0251] 2. 48-192 most redundant cDNA clones from the same library basedon previous sequencing data.

[0252] 3. 192 most redundant cDNA clones in the entire maize sequencedatabase.

[0253] 4. A Sal-A20 oligo nucleotide of SEQ ID NO 3: TCG ACC CAC GCG TCCGAA AAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail butno cDNA.

[0254] 5. cDNA clones derived from rRNA.

[0255] The image of the autoradiography is scanned into computer and thesignal intensity and cold colony addresses of each colony is analyzed.Re-arraying of cold-colonies from 384 well plates to 96 well plates isconducted using Q-bot.

EXAMPLE 4

[0256] This example describes identification of the gene from a computerhomology search.

[0257] Gene identities can be determined by conducting BLAST (BasicLocal Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol.Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences are analyzed forsimilarity to all publicly available DNA sequences contained in the “nr”database using the BLASTN algorithm. The DNA sequences are translated inall reading frames and compared for similarity to all publicly availableprotein sequences contained in the “nr” database using the BLASTXalgorithm (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993))provided by the NCBI. In some cases, the sequencing data from two ormore clones containing overlapping segments of DNA are used to constructcontiguous DNA sequences.

[0258] Sequence alignments and percent identity calculations can beperformed using the Megalign program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences can be performed using the Clustal method of alignment(Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters(GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments using the Clustal method are KTUPLE 1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5.

EXAMPLE 5

[0259] This example describes expression of transgenes in monocot cells.

[0260] A transgene comprising a cDNA encoding the instant polypeptidesin sense orientation with respect to the maize 27 kD zein promoter thatis located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or SmaI) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL1-Blue (EpicurianColi XL-1 Blue; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (SequenaseDNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a transgene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

[0261] The transgene described above can then be introduced into corncells by the following procedure. Immature corn embryos can be dissectedfrom developing caryopses derived from crosses of the inbred corn linesH99 and LH132. The embryos are isolated 10 to 11 days after pollinationwhen they are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

[0262] The plasmid, p35S/Ac (Hoechst Ag, Frankfurt, Germany) orequivalent may be used in transformation experiments in order to providefor a selectable marker. This plasmid contains the Pat gene (seeEuropean Patent Publication 0 242 236) which encodes phosphinothricinacetyl transferase (PAT). The enzyme PAT confers resistance toherbicidal glutamine synthetase inhibitors such as phosphinothricin. Thepat gene in p35S/Ac is under the control of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

[0263] The particle bombardment method (Klein et al. (1987) Nature327:70-73) may be used to transfer genes to the callus culture cells.According to this method, gold particles (1 μm in diameter) are coatedwith DNA using the following technique. Ten μg of plasmid DNAs are addedto 50 μL of a suspension of gold particles (60 mg per mL). Calciumchloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL ofa 1.0 M solution) are added to the particles. The suspension is vortexedduring the addition of these solutions. After 10 minutes, the tubes arebriefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.The particles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules, Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

[0264] For bombardment, the embryogenic tissue is placed on filter paperover agarose-solidified N6 medium. The tissue is arranged as a thin lawnand covered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

[0265] Seven days after bombardment the tissue can be transferred to N6medium that contains glufosinate (2 mg per liter) and lacks casein orproline. The tissue continues to grow slowly on this medium. After anadditional 2 weeks the tissue can be transferred to fresh N6 mediumcontaining glufosinate. After 6 weeks, areas of about 1 cm in diameterof actively growing callus can be identified on some of the platescontaining the glufosinate-supplemented medium. These calli may continueto grow when sub-cultured on the selective medium.

[0266] Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

EXAMPLE 6

[0267] This example describes expression of transgenes in dicot cells.

[0268] A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

[0269] The cDNA fragment of this gene may be generated by polymerasechain reaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

[0270] Soybean embryos may then be transformed with the expressionvector comprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

[0271] Soybean embryogenic suspension cultures can maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

[0272] Soybean embryogenic suspension cultures may then be transformedby the method of particle gun bombardment (Klein et al. (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

[0273] A selectable marker gene which can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et a/.(1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

[0274] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

[0275] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue are normally bombarded. Membranerupture pressure is set at 1100 psi and the chamber is evacuated to avacuum of 28 inches mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue can be divided in half and placed backinto liquid and cultured as described above.

[0276] Five to seven days post bombardment, the liquid media may beexchanged with fresh media, and eleven to twelve days post bombardmentwith fresh media containing 50 mg/mL hygromycin. This selective mediacan be refreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 7

[0277] This example describes expression of a transgene in microbialcells.

[0278] The cDNAs encoding the instant polypeptides can be inserted intothe T7 E. coli expression vector pBT430. This vector is a derivative ofpET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0279] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

[0280] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J.Mol. Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 h at 25°. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One microgramof protein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

EXAMPLE 8 Determination of Effect of Mutator (Mu) Insertion into Sus1Gene

[0281] This example describes the procedure to identify plantscontaining Mu inserted into constitutive sucrose synthase gene, andphenotypic and biochemical analyses of the mutant plants.

[0282] The Trait Utility System for Corn (TUSC; see U.S. Pat. No.5,962,764) is a method that employs genetic and molecular techniques tofacilitate the study of gene function in maize. Studying gene functionimplies that the gene's sequence is already known, thus the method worksin reverse: from sequence to phenotype. This kind of application isreferred to as “reverse genetics”, which contrasts with “forward”methods (such as transposon tagging) that are designed to identify andisolate the gene(s) responsible for a particular trait (phenotype).

[0283] Pioneer Hi-Bred International, Inc., has a proprietary collectionof maize genomic DNA from approximately 42,000 individual F. plants(Reverse genetics for maize., Meeley, R and Briggs, S, 1995, MaizeGenet. Coop. Newslett. 69:67,82). The genome of each of theseindividuals contains multiple copies of the transposable element family,Mutator (Mu). The Mu family is highly mutagenic; in the presence of theactive element Mu-DR, these elements transpose throughout the genome,inserting into genic regions, and often disrupting gene function. Bycollecting genomic DNA from a large number of individuals, Pioneer hasassembled a library of the mutagenized maize genome. Mu insertion eventsare predominately heterozygous so, given the recessive nature of mostinsertional mutations, the F₁ plants appear wild-type. Each of theplants was selfed to produce F₂ seed, which was collected. In generatingthe F₂ progeny, insertional mutations segregate in a Mendelian fashionso are useful for investigating a mutant allele's effect on thephenotype. The TUSC system has been successfully used by a number oflaboratories to identify the function of a variety of genes (Cloning andcharacterization of the maize An1 gene, Bensen, R. J. et al., 1995,Plant Cell 7:75-84; Diversification of C-function activity in maizeflower development, Mena, M. et al., 1996, Science 274:1537-1540;Analysis of a chemical plant defense mechanism in grasses, Frey, M. etal., 1997, Science 277:696-699; The control of maize spikelet meristemfate by the APETALA2-like gene Indeterminate spikelet 1, Chuck, G., etal., 1998, Genes & Development 12:1145-1154; A SecY homologue isrequired for the elaboration of the chloroplast thylakoid membrane andfor normal chloroplast gene expression, Roy, L. M. et al., 1998, J. CellBiol. 141:1-11).

[0284] PCR Screening for Mu insertions in Sus1:

[0285] Two primers were designed from within the Sus1 cDNA anddesignated as gene-specific primers (GSPs): Forward primer (GSP1) of SEQID NO:8: 5′-ACGGAATCGTTCGCAAGTGGATCTC-3′ Reverse primer (GSP2) of SEQ IDNO:9: 5′-GATGATTGGCTTGTTCCTGTCGTTC-3′

[0286] These primers are about 1 kb apart with respect to the genomicsequence of Sus1.

[0287] Mu TIR primer of SEQ ID NO:10

[0288] 5′-AGA GM GCC MC GCC AWC GCC TCY ATT TCG TC-3′

[0289] To select primers for PCR we used Pickoligo. This program choosesthe T_(m) according to the following equation:

Tm=[((GC*3+AT*2)*37−562)/length]−5

[0290] PCR reactions were run with an annealing temperature of 62 C anda thermocycling profile as follows:$\underset{\_}{{94\quad}^{\circ}{C.\quad {- \quad 2^{\prime}}}}\left( {{initial}\quad ({denaturation})\begin{matrix}{\quad/} & {\quad {{94\quad}^{\circ}{C.\quad {- \quad 30^{''}}}\text{-}1^{\prime}}} & \quad \\{35\quad {cycles}} & {\quad {{62\quad}^{\circ}{C.\quad {- \quad 30^{''}}}\text{-}2^{\prime}}} & \quad \\{\quad \backslash} & \underset{\_}{{72\quad}^{\circ}{C.\quad {- \quad 1}}\text{-}3^{\prime}} & \quad \\\quad & {\quad {{72\quad}^{\circ}{C.\quad {- \quad 5^{\prime}}}}} & \left( {{final}\quad {extension}} \right)\end{matrix}} \right.$

[0291] Gel electrophoresis of the PCR products confirmed that there wasno false priming in single primer reactions and that only one fragmentwas amplified in paired GSP reactions.

[0292] The genomic DNA from 42,000 plants, combined into pools of 48plants each, was subjected to PCR with either GSP1 or GSP2 and Mu TIR.The pools that were confirmed to be positive by dot-blot hybridizationusing Sus1 cDNA as a probe were subjected to gel-blot analysis in orderto determine the size of fragments amplified. The pools in which cleanfragments were identified were subjected to further analysis to identifythe individual plants within those pools that contained Mu insertion(s).

[0293] Seed from F₁ plants identified in this manner was planted in thefield. Leaf discs from twenty plants from each F₂ row were collected andgenomic DNA isolated. The same twenty plants were selfed and the F₃ seedsaved. Pooled DNA (from 20 plants) from each of the twelve rows wassubjected to PCR using GSP1 or GSP2 and Mu TIR primer as mentionedabove. Three pools identified to contain Mu insertions were subjected toindividual plant analysis and homozygotes identified. The PCR-amplifiedfragments were cloned into TOPO vector (Invitrogen) and sequenced. TheMu insertion sites were determined by comparing the sequences obtainedwith the Sus1 and Mu sequences and are presented in FIG. 6, along withthe surrounding signature sequences. Both the insertions are within 3nucleotides of each other in the open reading frame corresponding to the12^(th) exon, suggesting that this region in the gene might represent ahot spot for Mu insertion.

[0294] From the stalks of homozygous mutant plants and their wildtypesibs, two internodes subtending the ear node were collected about twoweeks before final harvest. Cellulose and lignin concentrations weredetermined on the ground samples from the dried internodes. Theconcentration of cellulose is 30% less in the mutant plants than intheir wild-type sibs when considered as a percentage of total drymatter, and 6% less as a percentage of structural dry matter (FIG. 7).Significant reduction is also observed for structural dry matter in themutant plants. This is consistent with the hypothesis that UDP-glucosederived from the action of sucrose synthase plays a significant role incellulose biosynthesis. It also appears that a reduction in celluloseproduction adversely affects cell wall formation.

EXAMPLE 9 Alignment of Sucrose Synthase Amino Acid Sequences Includingthat of SUS3

[0295] Alignment was performed using AlignX program from Vector NTI. SH1and SUS1 are about 80% identical and about 90% similar. SUS3 is about70% identical and 80% similar to both SH1 and SUS1 (see FIG. 8).Sufficient differences exist with respect to both SH1 and SUS1 as toclassify SUS3 as a different protein. Since the short arm of chromosome1 is considered to be a duplication of the long arm of chromosome 9, themap location of Sus3 (bin 1.04) implies that it might be ancestrallyrelated to Sus1 (map location 9.05). However, based on homologyanalysis, it appears to have evolved independently of Sh1 and Sus1. Likeevolution of sucrose synthase genes apparently is chromosome-dependent,as Sh1 and Sus1, both on chromosome 9, share significantly greatersimilarity than does either of these with Sus3, although Sus3 isapparently a duplication of Sus1.

[0296] SUS3 in SEQ ID NO 1 appears to be missing 5-10 amino acids at theN-terminal end. Predicted molecular mass of the slightly truncated SUS3,802 amino acids long, is 91 kDa. The molecular mass might be adjusted0.5-1 kDa upward once the full-length cDNA is isolated. The predictedmolecular masses of SH1 and SUS1 are 91.7 and 92.8 kDa, respectively.Respective isolectric points (pl) of SUS3, SH1, and SUS1 are: 6.07,5.96, and 6.04.

EXAMPLE 10 Multiple Alignment of Maize Sucrose Synthase PolynucleotidesIncluding that of ZmSus3

[0297] The alignment was performed using the AlignX program of theVector NTI suite. Sh1 and Sus1 are 67% similar; either of these genes isabout 60% similar to Sus3 (see FIG. 9). A similar trend was observed atthe amino acid sequence level (see FIG. 8 and Example 9).

EXAMPLE 11 Polynucleotide and Polypeptide Encoding Deduced Full LengthSus3 using SEQ ID NO: 1 and Sorghum Sequence (SEQ ID NO: 13) forCompletion of N-Terminal End

[0298] Sequencing of a complete full length native Zea mays cDNA whichencodes for the full length Sus3 has not been possible to date due tothe low expression level of Sus3 in maize and corresponding lowrepresentation in maize cDNA libraries. However, a sorghum EST of about345 nucleotides, GenBank Accession No. BF481989 (SEQ ID NO: 13), shows ahigh level of homology at the 3′ end to the 5′ end of SEQ ID NO.1. Byaligning SEQ ID NO: 1 and SEQ ID NO: 13 (FIG. 10 and FIG. 11), it waspossible to locate the ATG encoding the first methionine in SEQ ID NO:13 and determine an open reading frame through and into the aligned SEQID NO: 1. The inclusion of this short segment of sorghum sequence fromSEQ ID NO: 13 with the predominantly full length cDNA sequence of Sus3of SEQ ID NO: 1 provides the deduced full length polynucleotide of theSus3 (SEQ ID NO: 11) which encodes the full length Sus3 polypeptide (SEQID NO: 12). Thus this sorghum-maize hybrid sequence was used to supplythe deduced N-terminal end of the deduced full length Sus3 protein (SEQID NO: 12).

EXAMPLE 12 Determination of Polynucleotide Encoding Full Length Zea maysSus3 using Genomic DNA

[0299] Sequencing of a complete full length native Zea mays Sus3 can beaccomplished by using SEQ ID NO. 11 or SEQ ID NO: 13 to identify forisolation the fragment of genomic Zea mays DNA encoding untranslatedregion and 5′ end of the Zea mays Sus3 gene. This isolated genomicfragment is then sequences and the exons identified to verify the maizeSus3 cDNA sequence.

[0300] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited are hereby incorporated by reference.

1 13 1 2737 DNA Zea mays 1 gtcgacccac gcgtccggcg accgcgtcga ggacaccctccacgcgcacc gcaacgagct 60 cgtcgccctc ctgtccaagt acgtgaacaa ggggaagggcatcctgcagc cgcaccacat 120 cctcgacgcg ctcgacgagg tccagggctc cgggggccgcgcgctagccg agggaccctt 180 cctcgacgtc ctccgctccg cgcaggaggc gatcgtgctgccgccgttcg tggccatcgc 240 ggtgcgcccg cgcccgggag tttgggagta cgtccgcgtcaacgttcacg agctcagcgt 300 cgagcagctc acagtctcgg agtacctccg cttcaaggaggagcttgtcg acggccagca 360 caatgatccc tacgttctcg agcttgactt cgagccgttcaatgtctcag tcccacgccc 420 aaatcggtca tcatctattg gaaacggtgt gcagttcctcaaccgacact tgtcctcaat 480 catgttccgc aacagggatt gcttggagcc cctgttggatttcctccgtg gccaccggca 540 caaggggcat gttatgatgc ttaatgatag aatacaaagcttggggaggc ttcagtctgt 600 gctgaccaaa gctgaggagc acttgtcaaa gctccctgctgacacaccat actcacaatt 660 tgcttataaa tttcaagagt ggggcctgga gaaaggttggggtgatacag caggacatgt 720 tttggaaatg atccatctcc ttctagacat cattcaggcgccagacccat ctaccctaga 780 gaaattcttg gggaggatcc ccatgatttt taacgttgttgtggtatccc ctcatggata 840 ctttggtcaa gctaatgtat taggcttgcc agacacaggaggacagatcg tctatatact 900 ggaccaagtc cgtgcactag aaaatgagat ggttctccgtttaaagaaac aagggcttga 960 tgtttcccca aagattctca ttgttactcg gctgataccagatgcaaaag gaacatcatg 1020 caatcagcgg cttgagagaa ttagtggaac acagcatacttacatattac gagttccctt 1080 cagaaatgaa aatgggatac ttaagaaatg gatatcaagatttgatgtgt ggccatatct 1140 ggaaacattt gctgaggatg ctgctggtga aattgctgctgaattacaag gtactccaga 1200 cttcataatt ggaaactaca gtgatggaaa tcttgtggcgtcattgctat cttacaagat 1260 gggaattacc cagtgcaaca ttgctcatgc tctggaaaagactaagtatc cagattcaga 1320 catattttgg aagaatttcg atgagaagta ccatttctcctgccagttca ctgctgatat 1380 aattgctatg aacaatgctg attttatcat caccagcacataccaagaaa ttgctggaag 1440 caaaaatact gttggacagt atgagagtca tactgcctttactctgcctg gtctgtaccg 1500 agttgtccat gggatcgatg tcttcgatcc aaagttcaatatagtctctc ctggagctga 1560 catgtccata tactttccac ataccgagaa ggccaagcgactcacctctc ttcatggttc 1620 aatcgaaaat ttgatttatg acccggagca aaacgatgaacacattgggc atctggatga 1680 ccggtcaaag cccatcctct tctccatggc aagactcgacagggtgaaga acataacagg 1740 gctggtcgaa gcttttgcta agtgcgctaa gctgagggagctggtaaacc ttgtcgtcgt 1800 tgccgggtac aatgatgtca acaagtccaa ggacagggaagagatcgcgg agatagagaa 1860 gatgcatgaa ctcatcaaga cccacaactt gttcgggcagttccgctgga tctctgccca 1920 gacaaacagg gcccgtaacg gcgagctcta tcgctacatcgctgataccc atggtgcttt 1980 cgtacagccg gccttgtatg aagcgttcgg tctcaccgtcgttgaggcca tgacctgtgg 2040 gcttcctact ttcgcgacgc tccatggagg tccagctgagatcatagagc atggcgtctc 2100 gggcttccac attgacccgt accaccccga acaggctgttaatctgatgg ccgacttctt 2160 cgaccggtgc aagcaagacc cagatcactg ggtgaatatatctggagcag ggctgcagcg 2220 catatacgag aagtacacat ggaagatata ctcagagaggttgatgacac tggccggggt 2280 ctacggtttc tggaagtacg tgtcgaagct cgagaggctggagacgaggc gctaccttga 2340 gatgttctac atactgaagt tccgcgagct ggcgaagaccgtgccgcttg caattgacca 2400 accgcagtag cttgcgcaac tgcgactgcg tagcacttggtacaagactg aaacctgaag 2460 gaccttcagt aatttaggcg cggcagacgg tagccaataaaatgtgccgg agctgaactg 2520 gttttttatt atgtacataa tggcagtata acaaaattactgaaggcagg tgggttgcag 2580 ttgtgtgttc gttactgttt actgtattat gtcaagctgtcggctgcaat ttctttgctg 2640 gcaagccgca ggcactggtg aagtgctgat aaatacatcatattctgttg acctgtgaaa 2700 aaaaaaaaaa aaaaaaaaaa aaaaaaaggg cggccgc 27372 802 PRT Zea mays 2 Ser Thr His Ala Ser Gly Asp Arg Val Glu Asp Thr LeuHis Ala His 1 5 10 15 Arg Asn Glu Leu Val Ala Leu Leu Ser Lys Tyr ValAsn Lys Gly Lys 20 25 30 Gly Ile Leu Gln Pro His His Ile Leu Asp Ala LeuAsp Glu Val Gln 35 40 45 Gly Ser Gly Gly Arg Ala Leu Ala Glu Gly Pro PheLeu Asp Val Leu 50 55 60 Arg Ser Ala Gln Glu Ala Ile Val Leu Pro Pro PheVal Ala Ile Ala 65 70 75 80 Val Arg Pro Arg Pro Gly Val Trp Glu Tyr ValArg Val Asn Val His 85 90 95 Glu Leu Ser Val Glu Gln Leu Thr Val Ser GluTyr Leu Arg Phe Lys 100 105 110 Glu Glu Leu Val Asp Gly Gln His Asn AspPro Tyr Val Leu Glu Leu 115 120 125 Asp Phe Glu Pro Phe Asn Val Ser ValPro Arg Pro Asn Arg Ser Ser 130 135 140 Ser Ile Gly Asn Gly Val Gln PheLeu Asn Arg His Leu Ser Ser Ile 145 150 155 160 Met Phe Arg Asn Arg AspCys Leu Glu Pro Leu Leu Asp Phe Leu Arg 165 170 175 Gly His Arg His LysGly His Val Met Met Leu Asn Asp Arg Ile Gln 180 185 190 Ser Leu Gly ArgLeu Gln Ser Val Leu Thr Lys Ala Glu Glu His Leu 195 200 205 Ser Lys LeuPro Ala Asp Thr Pro Tyr Ser Gln Phe Ala Tyr Lys Phe 210 215 220 Gln GluTrp Gly Leu Glu Lys Gly Trp Gly Asp Thr Ala Gly His Val 225 230 235 240Leu Glu Met Ile His Leu Leu Leu Asp Ile Ile Gln Ala Pro Asp Pro 245 250255 Ser Thr Leu Glu Lys Phe Leu Gly Arg Ile Pro Met Ile Phe Asn Val 260265 270 Val Val Val Ser Pro His Gly Tyr Phe Gly Gln Ala Asn Val Leu Gly275 280 285 Leu Pro Asp Thr Gly Gly Gln Ile Val Tyr Ile Leu Asp Gln ValArg 290 295 300 Ala Leu Glu Asn Glu Met Val Leu Arg Leu Lys Lys Gln GlyLeu Asp 305 310 315 320 Val Ser Pro Lys Ile Leu Ile Val Thr Arg Leu IlePro Asp Ala Lys 325 330 335 Gly Thr Ser Cys Asn Gln Arg Leu Glu Arg IleSer Gly Thr Gln His 340 345 350 Thr Tyr Ile Leu Arg Val Pro Phe Arg AsnGlu Asn Gly Ile Leu Lys 355 360 365 Lys Trp Ile Ser Arg Phe Asp Val TrpPro Tyr Leu Glu Thr Phe Ala 370 375 380 Glu Asp Ala Ala Gly Glu Ile AlaAla Glu Leu Gln Gly Thr Pro Asp 385 390 395 400 Phe Ile Ile Gly Asn TyrSer Asp Gly Asn Leu Val Ala Ser Leu Leu 405 410 415 Ser Tyr Lys Met GlyIle Thr Gln Cys Asn Ile Ala His Ala Leu Glu 420 425 430 Lys Thr Lys TyrPro Asp Ser Asp Ile Phe Trp Lys Asn Phe Asp Glu 435 440 445 Lys Tyr HisPhe Ser Cys Gln Phe Thr Ala Asp Ile Ile Ala Met Asn 450 455 460 Asn AlaAsp Phe Ile Ile Thr Ser Thr Tyr Gln Glu Ile Ala Gly Ser 465 470 475 480Lys Asn Thr Val Gly Gln Tyr Glu Ser His Thr Ala Phe Thr Leu Pro 485 490495 Gly Leu Tyr Arg Val Val His Gly Ile Asp Val Phe Asp Pro Lys Phe 500505 510 Asn Ile Val Ser Pro Gly Ala Asp Met Ser Ile Tyr Phe Pro His Thr515 520 525 Glu Lys Ala Lys Arg Leu Thr Ser Leu His Gly Ser Ile Glu AsnLeu 530 535 540 Ile Tyr Asp Pro Glu Gln Asn Asp Glu His Ile Gly His LeuAsp Asp 545 550 555 560 Arg Ser Lys Pro Ile Leu Phe Ser Met Ala Arg LeuAsp Arg Val Lys 565 570 575 Asn Ile Thr Gly Leu Val Glu Ala Phe Ala LysCys Ala Lys Leu Arg 580 585 590 Glu Leu Val Asn Leu Val Val Val Ala GlyTyr Asn Asp Val Asn Lys 595 600 605 Ser Lys Asp Arg Glu Glu Ile Ala GluIle Glu Lys Met His Glu Leu 610 615 620 Ile Lys Thr His Asn Leu Phe GlyGln Phe Arg Trp Ile Ser Ala Gln 625 630 635 640 Thr Asn Arg Ala Arg AsnGly Glu Leu Tyr Arg Tyr Ile Ala Asp Thr 645 650 655 His Gly Ala Phe ValGln Pro Ala Leu Tyr Glu Ala Phe Gly Leu Thr 660 665 670 Val Val Glu AlaMet Thr Cys Gly Leu Pro Thr Phe Ala Thr Leu His 675 680 685 Gly Gly ProAla Glu Ile Ile Glu His Gly Val Ser Gly Phe His Ile 690 695 700 Asp ProTyr His Pro Glu Gln Ala Val Asn Leu Met Ala Asp Phe Phe 705 710 715 720Asp Arg Cys Lys Gln Asp Pro Asp His Trp Val Asn Ile Ser Gly Ala 725 730735 Gly Leu Gln Arg Ile Tyr Glu Lys Tyr Thr Trp Lys Ile Tyr Ser Glu 740745 750 Arg Leu Met Thr Leu Ala Gly Val Tyr Gly Phe Trp Lys Tyr Val Ser755 760 765 Lys Leu Glu Arg Leu Glu Thr Arg Arg Tyr Leu Glu Met Phe TyrIle 770 775 780 Leu Lys Phe Arg Glu Leu Ala Lys Thr Val Pro Leu Ala IleAsp Gln 785 790 795 800 Pro Gln 3 36 DNA Artificial Sequence Designedoligonucleotide based upon the adapter sequence and poly T to removeclones which have a poly A tail but no cDNA. 3 tcgacccacg cgtccgaaaaaaaaaaaaaa aaaaaa 36 4 2746 DNA Zea mays CDS (72)...(2480) 4 aaaccctccctccctcctcc attggactgc ttgctccctg ttgaccattg ggtattctga 60 accatcgagc catg gct gcc aag ctg act cgc ctt cac agt ctt cgc gaa 110 Met Ala Ala LysLeu Thr Arg Leu His Ser Leu Arg Glu 1 5 10 cgc ctt ggt gcc acc ttc tcctcc cat ccc aat gaa ctg ata gca ctc 158 Arg Leu Gly Ala Thr Phe Ser SerHis Pro Asn Glu Leu Ile Ala Leu 15 20 25 ttt tcc agg tat gtt cac cag ggcaag gga atg ctt cag cgc cat cag 206 Phe Ser Arg Tyr Val His Gln Gly LysGly Met Leu Gln Arg His Gln 30 35 40 45 ctg ctt gcg gag ttt gat gcc ctgttt gat agt gac aag gag aag tat 254 Leu Leu Ala Glu Phe Asp Ala Leu PheAsp Ser Asp Lys Glu Lys Tyr 50 55 60 gca cca ttt gaa gac att ctt cgt gctgct cag gaa gca att gtg ctc 302 Ala Pro Phe Glu Asp Ile Leu Arg Ala AlaGln Glu Ala Ile Val Leu 65 70 75 ccc cca tgg gtt gca ctt gct atc agg ccaagg cct ggt gtc tgg gat 350 Pro Pro Trp Val Ala Leu Ala Ile Arg Pro ArgPro Gly Val Trp Asp 80 85 90 tac att cgg gtg aat gta agt gag ctg gct gtggag gag ctg agt gtt 398 Tyr Ile Arg Val Asn Val Ser Glu Leu Ala Val GluGlu Leu Ser Val 95 100 105 tct gag tac ttg gca ttc aag gaa cag ctg gtggat gga caa tcc aac 446 Ser Glu Tyr Leu Ala Phe Lys Glu Gln Leu Val AspGly Gln Ser Asn 110 115 120 125 agc aac ttt gtg ctt gag ctt gat ttt gagccc ttc aat gcc tcc ttt 494 Ser Asn Phe Val Leu Glu Leu Asp Phe Glu ProPhe Asn Ala Ser Phe 130 135 140 cct cgt cct tcc atg tcg aag tcc atc ggaaat gga gtg caa ttc ctt 542 Pro Arg Pro Ser Met Ser Lys Ser Ile Gly AsnGly Val Gln Phe Leu 145 150 155 aac cga cac ctg tcg tcc aag ttg ttc caggac aag gag agt ttg tac 590 Asn Arg His Leu Ser Ser Lys Leu Phe Gln AspLys Glu Ser Leu Tyr 160 165 170 ccc ttg ctg aac ttc ctc aag gct cat aactac aag ggc acg acg atg 638 Pro Leu Leu Asn Phe Leu Lys Ala His Asn TyrLys Gly Thr Thr Met 175 180 185 atg ttg aat gac aga atc caa agc ctt cgtggt ctc caa tca tcc ctg 686 Met Leu Asn Asp Arg Ile Gln Ser Leu Arg GlyLeu Gln Ser Ser Leu 190 195 200 205 aga aag gca gag gag tat cta ctg agtgtt cct caa gac act ccc tac 734 Arg Lys Ala Glu Glu Tyr Leu Leu Ser ValPro Gln Asp Thr Pro Tyr 210 215 220 tcg gag ttc aac cat agg ttc caa gagctt ggc ttg gag aag ggt tgg 782 Ser Glu Phe Asn His Arg Phe Gln Glu LeuGly Leu Glu Lys Gly Trp 225 230 235 ggt gac act gcg aag cgt gtt ctc gacaca ctc cac ttg ctt ctc gac 830 Gly Asp Thr Ala Lys Arg Val Leu Asp ThrLeu His Leu Leu Leu Asp 240 245 250 ctt ctt gag gcc cct gat cct gcc aacttg gag aag ttc ctt gga act 878 Leu Leu Glu Ala Pro Asp Pro Ala Asn LeuGlu Lys Phe Leu Gly Thr 255 260 265 ata cca atg atg ttc aac gtt gtt atcctg tct cct cat ggc tac ttc 926 Ile Pro Met Met Phe Asn Val Val Ile LeuSer Pro His Gly Tyr Phe 270 275 280 285 gcc cag tcc aat gtg ctt gga taccct gac act ggc ggt cag gtt gtg 974 Ala Gln Ser Asn Val Leu Gly Tyr ProAsp Thr Gly Gly Gln Val Val 290 295 300 tac att ctg gat caa gtc cgt gctttg gag aat gag atg ctt ctg agg 1022 Tyr Ile Leu Asp Gln Val Arg Ala LeuGlu Asn Glu Met Leu Leu Arg 305 310 315 att aag cag caa ggc ctt gat atcact ccg aag atc ctc att gtt acc 1070 Ile Lys Gln Gln Gly Leu Asp Ile ThrPro Lys Ile Leu Ile Val Thr 320 325 330 agg ctg ttg cct gat gct gct gggact acg tgc ggt cag cgg ctg gag 1118 Arg Leu Leu Pro Asp Ala Ala Gly ThrThr Cys Gly Gln Arg Leu Glu 335 340 345 aag gtc att ggt act gag cac acagac atc att cgc gtt ccc ttc aga 1166 Lys Val Ile Gly Thr Glu His Thr AspIle Ile Arg Val Pro Phe Arg 350 355 360 365 aat gag aat ggc atc ctc cgcaag tgg atc tct cgt ttt gat gtc tgg 1214 Asn Glu Asn Gly Ile Leu Arg LysTrp Ile Ser Arg Phe Asp Val Trp 370 375 380 cca tac ctg gag aca tac actgag gat gtt tcc agt gaa ata atg aaa 1262 Pro Tyr Leu Glu Thr Tyr Thr GluAsp Val Ser Ser Glu Ile Met Lys 385 390 395 gaa atg cag gcc aag cct gacctt atc att ggc aac tac agc gat ggc 1310 Glu Met Gln Ala Lys Pro Asp LeuIle Ile Gly Asn Tyr Ser Asp Gly 400 405 410 aac cta gtc gcc act ctg ctcgcg cac aag ttg gga gtc act cag tgt 1358 Asn Leu Val Ala Thr Leu Leu AlaHis Lys Leu Gly Val Thr Gln Cys 415 420 425 acc atc gct cat gcc ttg gagaaa acc aaa tac ccc aac tcg gac atc 1406 Thr Ile Ala His Ala Leu Glu LysThr Lys Tyr Pro Asn Ser Asp Ile 430 435 440 445 tac ttg gac aaa ttc gacagc cag tac cac ttc tct tgc cag ttc aca 1454 Tyr Leu Asp Lys Phe Asp SerGln Tyr His Phe Ser Cys Gln Phe Thr 450 455 460 gct gac ctt att gcc atgaac cac acc gat ttc atc atc acc agc aca 1502 Ala Asp Leu Ile Ala Met AsnHis Thr Asp Phe Ile Ile Thr Ser Thr 465 470 475 ttc caa gaa atc gcg ggaagc aag gac acc gtg ggg cag tac gag tcc 1550 Phe Gln Glu Ile Ala Gly SerLys Asp Thr Val Gly Gln Tyr Glu Ser 480 485 490 cat atc gcg ttc act cttcct ggg ctc tac cgt gtc gtc cat ggc atc 1598 His Ile Ala Phe Thr Leu ProGly Leu Tyr Arg Val Val His Gly Ile 495 500 505 gat gtt ttc gat ccc aagttc aac att gtc tct cct gga gca gac atg 1646 Asp Val Phe Asp Pro Lys PheAsn Ile Val Ser Pro Gly Ala Asp Met 510 515 520 525 agt gtt tac tac ccttat acg gaa acc gac aag aga ctc act gcc ttc 1694 Ser Val Tyr Tyr Pro TyrThr Glu Thr Asp Lys Arg Leu Thr Ala Phe 530 535 540 cat cct gaa atc gaggag ctc atc tac agc gac gtc gag aac tcc gag 1742 His Pro Glu Ile Glu GluLeu Ile Tyr Ser Asp Val Glu Asn Ser Glu 545 550 555 cac aag ttc gtg ctgaag gac aag aag aag ccg atc atc ttc tcg atg 1790 His Lys Phe Val Leu LysAsp Lys Lys Lys Pro Ile Ile Phe Ser Met 560 565 570 gcg cgt ctc gac cgcgtg aag aac atg aca ggc ctg gtc gag atg tac 1838 Ala Arg Leu Asp Arg ValLys Asn Met Thr Gly Leu Val Glu Met Tyr 575 580 585 ggc aag aac gcg cgcctg agg gag ctg gcg aac ctc gtg atc gtt gcc 1886 Gly Lys Asn Ala Arg LeuArg Glu Leu Ala Asn Leu Val Ile Val Ala 590 595 600 605 ggt gac cac ggcaag gag tcc aag gac agg gag gag cag gcg gag ttc 1934 Gly Asp His Gly LysGlu Ser Lys Asp Arg Glu Glu Gln Ala Glu Phe 610 615 620 aag aag atg tacagc ctc atc gac gag tac aag ttg aag ggc cat atc 1982 Lys Lys Met Tyr SerLeu Ile Asp Glu Tyr Lys Leu Lys Gly His Ile 625 630 635 cgg tgg atc tcggcg cag atg aac cgt gtc cgc aac ggg gag ctg tac 2030 Arg Trp Ile Ser AlaGln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr 640 645 650 cgc tac att tgcgat acc aag ggc gca ttc gtg cag cct gcg ttc tac 2078 Arg Tyr Ile Cys AspThr Lys Gly Ala Phe Val Gln Pro Ala Phe Tyr 655 660 665 gaa gcg ttc ggcctg act gtg atc gag tcc atg acg tgc ggt ctg cca 2126 Glu Ala Phe Gly LeuThr Val Ile Glu Ser Met Thr Cys Gly Leu Pro 670 675 680 685 acg atc gcgacc tgc cat ggc ggc cct gct gag atc atc gtg gac ggg 2174 Thr Ile Ala ThrCys His Gly Gly Pro Ala Glu Ile Ile Val Asp Gly 690 695 700 gta tct ggcctg cac att gac cct tac cac agc gac aag gcc gcg gat 2222 Val Ser Gly LeuHis Ile Asp Pro Tyr His Ser Asp Lys Ala Ala Asp 705 710 715 atc ctg gtcaac ttc ttt gac aaa tgc aag gca gat ccg agc tac tgg 2270 Ile Leu Val AsnPhe Phe Asp Lys Cys Lys Ala Asp Pro Ser Tyr Trp 720 725 730 gac gag atctca cag ggc ggc ctg cag aga att tat gag aag tac acc 2318 Asp Glu Ile SerGln Gly Gly Leu Gln Arg Ile Tyr Glu Lys Tyr Thr 735 740 745 tgg aag ctctac tcc gag agg ctg atg acc ctg acc ggc gtg tac ggg 2366 Trp Lys Leu TyrSer Glu Arg Leu Met Thr Leu Thr Gly Val Tyr Gly 750 755 760 765 ttc tggaag tac gtg agc aac ctg gag agg cgc gag acc cgc cgc tac 2414 Phe Trp LysTyr Val Ser Asn Leu Glu Arg Arg Glu Thr Arg Arg Tyr 770 775 780 atc gagatg ttc tac gcc ctg aag tac cgt agc ctg gca agc cag gtt 2462 Ile Glu MetPhe Tyr Ala Leu Lys Tyr Arg Ser Leu Ala Ser Gln Val 785 790 795 ccg ctgtcc ttc gat tag tacggggaaa gaaggagaag aagaagaaga 2510 Pro Leu Ser PheAsp * 800 agcccaggcc ggagaaccat cgcctgcatt tcgatctgtt tcaccgcaattcgcattgtt 2570 agtcgtgtat tggagttatg tgtacttggt ttccaagaac tttggttccttctcgttttt 2630 tttccttgtt tgagcgtttt tgggcagcgc tggcctggtt cctagtatggtgggaattgg 2690 ctgcaccttt tgcttcgaat aaaaatgcct gctcgttcac ctgtcttccagagtgc 2746 5 802 PRT Zea mays 5 Met Ala Ala Lys Leu Thr Arg Leu His SerLeu Arg Glu Arg Leu Gly 1 5 10 15 Ala Thr Phe Ser Ser His Pro Asn GluLeu Ile Ala Leu Phe Ser Arg 20 25 30 Tyr Val His Gln Gly Lys Gly Met LeuGln Arg His Gln Leu Leu Ala 35 40 45 Glu Phe Asp Ala Leu Phe Asp Ser AspLys Glu Lys Tyr Ala Pro Phe 50 55 60 Glu Asp Ile Leu Arg Ala Ala Gln GluAla Ile Val Leu Pro Pro Trp 65 70 75 80 Val Ala Leu Ala Ile Arg Pro ArgPro Gly Val Trp Asp Tyr Ile Arg 85 90 95 Val Asn Val Ser Glu Leu Ala ValGlu Glu Leu Ser Val Ser Glu Tyr 100 105 110 Leu Ala Phe Lys Glu Gln LeuVal Asp Gly Gln Ser Asn Ser Asn Phe 115 120 125 Val Leu Glu Leu Asp PheGlu Pro Phe Asn Ala Ser Phe Pro Arg Pro 130 135 140 Ser Met Ser Lys SerIle Gly Asn Gly Val Gln Phe Leu Asn Arg His 145 150 155 160 Leu Ser SerLys Leu Phe Gln Asp Lys Glu Ser Leu Tyr Pro Leu Leu 165 170 175 Asn PheLeu Lys Ala His Asn Tyr Lys Gly Thr Thr Met Met Leu Asn 180 185 190 AspArg Ile Gln Ser Leu Arg Gly Leu Gln Ser Ser Leu Arg Lys Ala 195 200 205Glu Glu Tyr Leu Leu Ser Val Pro Gln Asp Thr Pro Tyr Ser Glu Phe 210 215220 Asn His Arg Phe Gln Glu Leu Gly Leu Glu Lys Gly Trp Gly Asp Thr 225230 235 240 Ala Lys Arg Val Leu Asp Thr Leu His Leu Leu Leu Asp Leu LeuGlu 245 250 255 Ala Pro Asp Pro Ala Asn Leu Glu Lys Phe Leu Gly Thr IlePro Met 260 265 270 Met Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr PheAla Gln Ser 275 280 285 Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln ValVal Tyr Ile Leu 290 295 300 Asp Gln Val Arg Ala Leu Glu Asn Glu Met LeuLeu Arg Ile Lys Gln 305 310 315 320 Gln Gly Leu Asp Ile Thr Pro Lys IleLeu Ile Val Thr Arg Leu Leu 325 330 335 Pro Asp Ala Ala Gly Thr Thr CysGly Gln Arg Leu Glu Lys Val Ile 340 345 350 Gly Thr Glu His Thr Asp IleIle Arg Val Pro Phe Arg Asn Glu Asn 355 360 365 Gly Ile Leu Arg Lys TrpIle Ser Arg Phe Asp Val Trp Pro Tyr Leu 370 375 380 Glu Thr Tyr Thr GluAsp Val Ser Ser Glu Ile Met Lys Glu Met Gln 385 390 395 400 Ala Lys ProAsp Leu Ile Ile Gly Asn Tyr Ser Asp Gly Asn Leu Val 405 410 415 Ala ThrLeu Leu Ala His Lys Leu Gly Val Thr Gln Cys Thr Ile Ala 420 425 430 HisAla Leu Glu Lys Thr Lys Tyr Pro Asn Ser Asp Ile Tyr Leu Asp 435 440 445Lys Phe Asp Ser Gln Tyr His Phe Ser Cys Gln Phe Thr Ala Asp Leu 450 455460 Ile Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr Phe Gln Glu 465470 475 480 Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser His IleAla 485 490 495 Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His Gly Ile AspVal Phe 500 505 510 Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp MetSer Val Tyr 515 520 525 Tyr Pro Tyr Thr Glu Thr Asp Lys Arg Leu Thr AlaPhe His Pro Glu 530 535 540 Ile Glu Glu Leu Ile Tyr Ser Asp Val Glu AsnSer Glu His Lys Phe 545 550 555 560 Val Leu Lys Asp Lys Lys Lys Pro IleIle Phe Ser Met Ala Arg Leu 565 570 575 Asp Arg Val Lys Asn Met Thr GlyLeu Val Glu Met Tyr Gly Lys Asn 580 585 590 Ala Arg Leu Arg Glu Leu AlaAsn Leu Val Ile Val Ala Gly Asp His 595 600 605 Gly Lys Glu Ser Lys AspArg Glu Glu Gln Ala Glu Phe Lys Lys Met 610 615 620 Tyr Ser Leu Ile AspGlu Tyr Lys Leu Lys Gly His Ile Arg Trp Ile 625 630 635 640 Ser Ala GlnMet Asn Arg Val Arg Asn Gly Glu Leu Tyr Arg Tyr Ile 645 650 655 Cys AspThr Lys Gly Ala Phe Val Gln Pro Ala Phe Tyr Glu Ala Phe 660 665 670 GlyLeu Thr Val Ile Glu Ser Met Thr Cys Gly Leu Pro Thr Ile Ala 675 680 685Thr Cys His Gly Gly Pro Ala Glu Ile Ile Val Asp Gly Val Ser Gly 690 695700 Leu His Ile Asp Pro Tyr His Ser Asp Lys Ala Ala Asp Ile Leu Val 705710 715 720 Asn Phe Phe Asp Lys Cys Lys Ala Asp Pro Ser Tyr Trp Asp GluIle 725 730 735 Ser Gln Gly Gly Leu Gln Arg Ile Tyr Glu Lys Tyr Thr TrpLys Leu 740 745 750 Tyr Ser Glu Arg Leu Met Thr Leu Thr Gly Val Tyr GlyPhe Trp Lys 755 760 765 Tyr Val Ser Asn Leu Glu Arg Arg Glu Thr Arg ArgTyr Ile Glu Met 770 775 780 Phe Tyr Ala Leu Lys Tyr Arg Ser Leu Ala SerGln Val Pro Leu Ser 785 790 795 800 Phe Asp 6 2908 DNA Zea mays CDS(28)...(2478) 6 gcctgaggat ccaggaagag gacagca atg ggg gaa ggt gca ggtgac cgt gtc 54 Met Gly Glu Gly Ala Gly Asp Arg Val 1 5 ctg agc cgc ctccac agc gtc agg gag cgc att ggc gac tca ctc tct 102 Leu Ser Arg Leu HisSer Val Arg Glu Arg Ile Gly Asp Ser Leu Ser 10 15 20 25 gcc cac ccc aatgag ctt gtc gcc gtc ttc acc agg ctg aaa aac ctt 150 Ala His Pro Asn GluLeu Val Ala Val Phe Thr Arg Leu Lys Asn Leu 30 35 40 gga aag ggt atg ctgcag ccc cac cag atc att gcc gag tac aac aat 198 Gly Lys Gly Met Leu GlnPro His Gln Ile Ile Ala Glu Tyr Asn Asn 45 50 55 gcg atc cct gag gct gagcgc gag aag ctc aag gat ggt gct ttt gag 246 Ala Ile Pro Glu Ala Glu ArgGlu Lys Leu Lys Asp Gly Ala Phe Glu 60 65 70 gat gtc ctg agg gca gct caggag gcg att gtc atc ccc cca tgg gtt 294 Asp Val Leu Arg Ala Ala Gln GluAla Ile Val Ile Pro Pro Trp Val 75 80 85 gca ctt gcc atc cgc cct agg cctggt gtc tgg gag tat gtg agg gtc 342 Ala Leu Ala Ile Arg Pro Arg Pro GlyVal Trp Glu Tyr Val Arg Val 90 95 100 105 aac gtc agt gag ctc gct gttgag gag ctg aga gtt cct gag tac ctg 390 Asn Val Ser Glu Leu Ala Val GluGlu Leu Arg Val Pro Glu Tyr Leu 110 115 120 cag ttc aag gaa cag ctt gtggaa gaa ggc ccc aac aac aac ttt gtt 438 Gln Phe Lys Glu Gln Leu Val GluGlu Gly Pro Asn Asn Asn Phe Val 125 130 135 ctt gag ctg gac ttt gag ccattc aat gcc tcc ttc ccc cgt cct tct 486 Leu Glu Leu Asp Phe Glu Pro PheAsn Ala Ser Phe Pro Arg Pro Ser 140 145 150 ctg tca aag tcc att ggc aatggc gtg cag ttc ctc aac agg cac ctg 534 Leu Ser Lys Ser Ile Gly Asn GlyVal Gln Phe Leu Asn Arg His Leu 155 160 165 tca tca aag ctc ttc cat gacaag gag agc atg tac ccc ttg ctc aac 582 Ser Ser Lys Leu Phe His Asp LysGlu Ser Met Tyr Pro Leu Leu Asn 170 175 180 185 ttc ctt cgc gcc cac aactac aag ggg atg acc atg atg ttg aac gac 630 Phe Leu Arg Ala His Asn TyrLys Gly Met Thr Met Met Leu Asn Asp 190 195 200 aga atc cgc agt ctc agtgct ctg caa ggt gcg ctg agg aag gct gag 678 Arg Ile Arg Ser Leu Ser AlaLeu Gln Gly Ala Leu Arg Lys Ala Glu 205 210 215 gag cac ctg tcc acc ctacaa gct gat acc cca tac tct gaa ttt cac 726 Glu His Leu Ser Thr Leu GlnAla Asp Thr Pro Tyr Ser Glu Phe His 220 225 230 cac agg ttc cag gaa cttggt ctg gag aag ggt tgg ggt gat tgc gct 774 His Arg Phe Gln Glu Leu GlyLeu Glu Lys Gly Trp Gly Asp Cys Ala 235 240 245 aag cgt gca cag gag actatc cac ctc ctc ttg gac ctc ctg gag gcc 822 Lys Arg Ala Gln Glu Thr IleHis Leu Leu Leu Asp Leu Leu Glu Ala 250 255 260 265 cca gat ccg tcc accctg gag aag ttc ctt gga acg atc ccc atg gtg 870 Pro Asp Pro Ser Thr LeuGlu Lys Phe Leu Gly Thr Ile Pro Met Val 270 275 280 ttc aat gtc gtt atcctc tcc cct cat ggt tac ttc gct caa gct aat 918 Phe Asn Val Val Ile LeuSer Pro His Gly Tyr Phe Ala Gln Ala Asn 285 290 295 gtc ttg ggt tac cctgac acc gga ggc cag gtt gtc tac atc ttg gat 966 Val Leu Gly Tyr Pro AspThr Gly Gly Gln Val Val Tyr Ile Leu Asp 300 305 310 caa gtg cgc gct atggag aac gaa atg ctg ctg agg atc aag cag tgt 1014 Gln Val Arg Ala Met GluAsn Glu Met Leu Leu Arg Ile Lys Gln Cys 315 320 325 ggt ctt gac atc acgccg aag atc ctt att gtc acc agg ttg ctc cct 1062 Gly Leu Asp Ile Thr ProLys Ile Leu Ile Val Thr Arg Leu Leu Pro 330 335 340 345 gat gca act ggcacc acc tgt ggc cag cgc ctt gag aag gtc ctt ggc 1110 Asp Ala Thr Gly ThrThr Cys Gly Gln Arg Leu Glu Lys Val Leu Gly 350 355 360 acc gag cac tgccat atc ctt cgc gtg cca ttc aga aca gaa aac gga 1158 Thr Glu His Cys HisIle Leu Arg Val Pro Phe Arg Thr Glu Asn Gly 365 370 375 atc gtt cgc aagtgg atc tcg cga ttt gaa gtc tgg ccg tac ctg gag 1206 Ile Val Arg Lys TrpIle Ser Arg Phe Glu Val Trp Pro Tyr Leu Glu 380 385 390 act tac act gatgac gtg gcg cat gag att gct gga gag ctt cag gcc 1254 Thr Tyr Thr Asp AspVal Ala His Glu Ile Ala Gly Glu Leu Gln Ala 395 400 405 aat cct gac ctgatc atc gga aac tac agt gac gga aac ctt gtt gcg 1302 Asn Pro Asp Leu IleIle Gly Asn Tyr Ser Asp Gly Asn Leu Val Ala 410 415 420 425 tgt ttg ctcgcc cac aag atg ggt gtt act cac tgt acc att gcc cat 1350 Cys Leu Leu AlaHis Lys Met Gly Val Thr His Cys Thr Ile Ala His 430 435 440 gcg ctt gagaaa act aag tac cct aac tcc gac ctc tac tgg aag aag 1398 Ala Leu Glu LysThr Lys Tyr Pro Asn Ser Asp Leu Tyr Trp Lys Lys 445 450 455 ttt gag gatcac tac cac ttc tcg tgc cag ttc acc act gac ttg att 1446 Phe Glu Asp HisTyr His Phe Ser Cys Gln Phe Thr Thr Asp Leu Ile 460 465 470 gca atg aaccat gcc gac ttc atc atc acc agt acc ttc caa gag atc 1494 Ala Met Asn HisAla Asp Phe Ile Ile Thr Ser Thr Phe Gln Glu Ile 475 480 485 gcc gga aacaag gac acc gtc ggc cag tac gag tca cac atg gcg ttc 1542 Ala Gly Asn LysAsp Thr Val Gly Gln Tyr Glu Ser His Met Ala Phe 490 495 500 505 aca atgcct ggc ctg tac cgc gtt gtc cac ggc att gat gtg ttc gac 1590 Thr Met ProGly Leu Tyr Arg Val Val His Gly Ile Asp Val Phe Asp 510 515 520 ccc aagttc aac atc gtg tct cct ggc gcg gac ctg tcc atc tac ttc 1638 Pro Lys PheAsn Ile Val Ser Pro Gly Ala Asp Leu Ser Ile Tyr Phe 525 530 535 ccg tacacc gag tcg cac aag agg ctg acc tcc ctt cac ccg gag att 1686 Pro Tyr ThrGlu Ser His Lys Arg Leu Thr Ser Leu His Pro Glu Ile 540 545 550 gag gagctc ctg tac agc caa acc gag aac acg gag cac aag ttc gtt 1734 Glu Glu LeuLeu Tyr Ser Gln Thr Glu Asn Thr Glu His Lys Phe Val 555 560 565 ctg aacgac agg aac aag cca atc atc ttc tcc atg gct cgt ctc gac 1782 Leu Asn AspArg Asn Lys Pro Ile Ile Phe Ser Met Ala Arg Leu Asp 570 575 580 585 cgtgtg aag aac ttg act ggg ctg gtg gag ctg tac ggc cgg aac aag 1830 Arg ValLys Asn Leu Thr Gly Leu Val Glu Leu Tyr Gly Arg Asn Lys 590 595 600 cggctg cag gag ctg gtg aac ctc gtg gtc gtc tgc ggc gac cat ggc 1878 Arg LeuGln Glu Leu Val Asn Leu Val Val Val Cys Gly Asp His Gly 605 610 615 aaccct tcc aag gac aag gag gag cag gcc gag ttc aag aag atg ttt 1926 Asn ProSer Lys Asp Lys Glu Glu Gln Ala Glu Phe Lys Lys Met Phe 620 625 630 gacctc atc gag cag tac aac ctg aac ggg cac atc cgc tgg atc tcc 1974 Asp LeuIle Glu Gln Tyr Asn Leu Asn Gly His Ile Arg Trp Ile Ser 635 640 645 gcccag atg aac cgc gtc cgc aac ggc gag ctg tac cgc tac atc tgc 2022 Ala GlnMet Asn Arg Val Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Cys 650 655 660 665gac acc aag ggc gcc ttc gtg cag cct gct ttc tac gag gct ttc ggg 2070 AspThr Lys Gly Ala Phe Val Gln Pro Ala Phe Tyr Glu Ala Phe Gly 670 675 680ctg acg gtg gtt gag gcc atg acc tgc ggc ctg ccc acg ttc gcc acc 2118 LeuThr Val Val Glu Ala Met Thr Cys Gly Leu Pro Thr Phe Ala Thr 685 690 695gcc tac ggc ggt ccg gcc gag atc atc gtg cac ggc gtg tct ggc tac 2166 AlaTyr Gly Gly Pro Ala Glu Ile Ile Val His Gly Val Ser Gly Tyr 700 705 710cac atc gac cct tac cag ggc gac aag gcg tcg gcc ctg ctc gtg gac 2214 HisIle Asp Pro Tyr Gln Gly Asp Lys Ala Ser Ala Leu Leu Val Asp 715 720 725ttc ttc gac aag tgc cag gcg gag ccg agc cac tgg agc aag atc tcc 2262 PhePhe Asp Lys Cys Gln Ala Glu Pro Ser His Trp Ser Lys Ile Ser 730 735 740745 cag ggc ggg ctc cag cgt atc gag gag aag tac acc tgg aag ctg tac 2310Gln Gly Gly Leu Gln Arg Ile Glu Glu Lys Tyr Thr Trp Lys Leu Tyr 750 755760 tcg gag agg ctg atg acc ctc acc ggc gtg tac ggg ttc tgg aag tac 2358Ser Glu Arg Leu Met Thr Leu Thr Gly Val Tyr Gly Phe Trp Lys Tyr 765 770775 gtg tcc aac ctg gag agg cgc gag acc cgg cgg tac ctg gag atg ctg 2406Val Ser Asn Leu Glu Arg Arg Glu Thr Arg Arg Tyr Leu Glu Met Leu 780 785790 tac gcg ctc aag tac cgc acc atg gcg agc acc gtg ccg ctg gcc gtg 2454Tyr Ala Leu Lys Tyr Arg Thr Met Ala Ser Thr Val Pro Leu Ala Val 795 800805 gag gga gag ccc tcc agc aag tga tgcgtgacgg cggccacaga cctgatcgat2508 Glu Gly Glu Pro Ser Ser Lys * 810 815 cgatgagcga gagggagcactcggagtgtc gtgtcttttc ccttgccatt tctttctttc 2568 ttctttttcc ttcccggaggcgaaaaaaaa agagtctgct tttgctaggc ggcgggcgtt 2628 cgttgctgct ctttgcttcaagagttaaaa tttacctacc ttgtcaaggt cttgttccat 2688 cattgatccg ggtgtcgcttgtagtagtct gatggactgt tagtagtttg cgttgcgtcg 2748 gttgagaggg aacgttggtggtggtggtgt gtgtgcagtc aggcgtggtg ctccctttgt 2808 ttcctggatg ggatgttgctccttgaataa taatcgtagt ggccttggag cccttttcct 2868 gaaataagag cagcatcctagtgcttacct ttgcagctgt 2908 7 816 PRT Zea mays 7 Met Gly Glu Gly Ala GlyAsp Arg Val Leu Ser Arg Leu His Ser Val 1 5 10 15 Arg Glu Arg Ile GlyAsp Ser Leu Ser Ala His Pro Asn Glu Leu Val 20 25 30 Ala Val Phe Thr ArgLeu Lys Asn Leu Gly Lys Gly Met Leu Gln Pro 35 40 45 His Gln Ile Ile AlaGlu Tyr Asn Asn Ala Ile Pro Glu Ala Glu Arg 50 55 60 Glu Lys Leu Lys AspGly Ala Phe Glu Asp Val Leu Arg Ala Ala Gln 65 70 75 80 Glu Ala Ile ValIle Pro Pro Trp Val Ala Leu Ala Ile Arg Pro Arg 85 90 95 Pro Gly Val TrpGlu Tyr Val Arg Val Asn Val Ser Glu Leu Ala Val 100 105 110 Glu Glu LeuArg Val Pro Glu Tyr Leu Gln Phe Lys Glu Gln Leu Val 115 120 125 Glu GluGly Pro Asn Asn Asn Phe Val Leu Glu Leu Asp Phe Glu Pro 130 135 140 PheAsn Ala Ser Phe Pro Arg Pro Ser Leu Ser Lys Ser Ile Gly Asn 145 150 155160 Gly Val Gln Phe Leu Asn Arg His Leu Ser Ser Lys Leu Phe His Asp 165170 175 Lys Glu Ser Met Tyr Pro Leu Leu Asn Phe Leu Arg Ala His Asn Tyr180 185 190 Lys Gly Met Thr Met Met Leu Asn Asp Arg Ile Arg Ser Leu SerAla 195 200 205 Leu Gln Gly Ala Leu Arg Lys Ala Glu Glu His Leu Ser ThrLeu Gln 210 215 220 Ala Asp Thr Pro Tyr Ser Glu Phe His His Arg Phe GlnGlu Leu Gly 225 230 235 240 Leu Glu Lys Gly Trp Gly Asp Cys Ala Lys ArgAla Gln Glu Thr Ile 245 250 255 His Leu Leu Leu Asp Leu Leu Glu Ala ProAsp Pro Ser Thr Leu Glu 260 265 270 Lys Phe Leu Gly Thr Ile Pro Met ValPhe Asn Val Val Ile Leu Ser 275 280 285 Pro His Gly Tyr Phe Ala Gln AlaAsn Val Leu Gly Tyr Pro Asp Thr 290 295 300 Gly Gly Gln Val Val Tyr IleLeu Asp Gln Val Arg Ala Met Glu Asn 305 310 315 320 Glu Met Leu Leu ArgIle Lys Gln Cys Gly Leu Asp Ile Thr Pro Lys 325 330 335 Ile Leu Ile ValThr Arg Leu Leu Pro Asp Ala Thr Gly Thr Thr Cys 340 345 350 Gly Gln ArgLeu Glu Lys Val Leu Gly Thr Glu His Cys His Ile Leu 355 360 365 Arg ValPro Phe Arg Thr Glu Asn Gly Ile Val Arg Lys Trp Ile Ser 370 375 380 ArgPhe Glu Val Trp Pro Tyr Leu Glu Thr Tyr Thr Asp Asp Val Ala 385 390 395400 His Glu Ile Ala Gly Glu Leu Gln Ala Asn Pro Asp Leu Ile Ile Gly 405410 415 Asn Tyr Ser Asp Gly Asn Leu Val Ala Cys Leu Leu Ala His Lys Met420 425 430 Gly Val Thr His Cys Thr Ile Ala His Ala Leu Glu Lys Thr LysTyr 435 440 445 Pro Asn Ser Asp Leu Tyr Trp Lys Lys Phe Glu Asp His TyrHis Phe 450 455 460 Ser Cys Gln Phe Thr Thr Asp Leu Ile Ala Met Asn HisAla Asp Phe 465 470 475 480 Ile Ile Thr Ser Thr Phe Gln Glu Ile Ala GlyAsn Lys Asp Thr Val 485 490 495 Gly Gln Tyr Glu Ser His Met Ala Phe ThrMet Pro Gly Leu Tyr Arg 500 505 510 Val Val His Gly Ile Asp Val Phe AspPro Lys Phe Asn Ile Val Ser 515 520 525 Pro Gly Ala Asp Leu Ser Ile TyrPhe Pro Tyr Thr Glu Ser His Lys 530 535 540 Arg Leu Thr Ser Leu His ProGlu Ile Glu Glu Leu Leu Tyr Ser Gln 545 550 555 560 Thr Glu Asn Thr GluHis Lys Phe Val Leu Asn Asp Arg Asn Lys Pro 565 570 575 Ile Ile Phe SerMet Ala Arg Leu Asp Arg Val Lys Asn Leu Thr Gly 580 585 590 Leu Val GluLeu Tyr Gly Arg Asn Lys Arg Leu Gln Glu Leu Val Asn 595 600 605 Leu ValVal Val Cys Gly Asp His Gly Asn Pro Ser Lys Asp Lys Glu 610 615 620 GluGln Ala Glu Phe Lys Lys Met Phe Asp Leu Ile Glu Gln Tyr Asn 625 630 635640 Leu Asn Gly His Ile Arg Trp Ile Ser Ala Gln Met Asn Arg Val Arg 645650 655 Asn Gly Glu Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val660 665 670 Gln Pro Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu AlaMet 675 680 685 Thr Cys Gly Leu Pro Thr Phe Ala Thr Ala Tyr Gly Gly ProAla Glu 690 695 700 Ile Ile Val His Gly Val Ser Gly Tyr His Ile Asp ProTyr Gln Gly 705 710 715 720 Asp Lys Ala Ser Ala Leu Leu Val Asp Phe PheAsp Lys Cys Gln Ala 725 730 735 Glu Pro Ser His Trp Ser Lys Ile Ser GlnGly Gly Leu Gln Arg Ile 740 745 750 Glu Glu Lys Tyr Thr Trp Lys Leu TyrSer Glu Arg Leu Met Thr Leu 755 760 765 Thr Gly Val Tyr Gly Phe Trp LysTyr Val Ser Asn Leu Glu Arg Arg 770 775 780 Glu Thr Arg Arg Tyr Leu GluMet Leu Tyr Ala Leu Lys Tyr Arg Thr 785 790 795 800 Met Ala Ser Thr ValPro Leu Ala Val Glu Gly Glu Pro Ser Ser Lys 805 810 815 8 25 DNA Zeamays 8 acggaatcgt tcgcaagtgg atctc 25 9 25 DNA Zea mays 9 gatgattggcttgttcctgt cgttc 25 10 32 DNA Zea mays 10 agagaagcca acgccawcgcctcyatttcg tc 32 11 2757 DNA Zea mays CDS (1)...(2430) source (1)...(39)Sorghum pronpinquum 11 atg tct gcc ccg aag ctg aac cgc aac gcg agc atccgg gac cgc gtc 48 Met Ser Ala Pro Lys Leu Asn Arg Asn Ala Ser Ile ArgAsp Arg Val 1 5 10 15 gag gac acc ctc cac gcg cac cgc aac gag ctc gtcgcc ctc ctc tcc 96 Glu Asp Thr Leu His Ala His Arg Asn Glu Leu Val AlaLeu Leu Ser 20 25 30 aag tac gtg aac aag ggg aag ggc atc ctg cag ccg caccac atc ctc 144 Lys Tyr Val Asn Lys Gly Lys Gly Ile Leu Gln Pro His HisIle Leu 35 40 45 gac gcg ctc gac gag gtc cag ggc tcc ggg gtc cgc gcg ctcgcc gag 192 Asp Ala Leu Asp Glu Val Gln Gly Ser Gly Val Arg Ala Leu AlaGlu 50 55 60 gga ccc ttc ctc gac gtc ctc cgc tcc gcg cag gag gcg atc gtgctg 240 Gly Pro Phe Leu Asp Val Leu Arg Ser Ala Gln Glu Ala Ile Val Leu65 70 75 80 ccg ccg ttc gtg gcc atc gcg gtg cgc ccg cgc ccg gga gtt tgggag 288 Pro Pro Phe Val Ala Ile Ala Val Arg Pro Arg Pro Gly Val Trp Glu85 90 95 tac gtc cgc gtc aac gtt cac gag ctc agc gtc gag cag ctc aca gtc336 Tyr Val Arg Val Asn Val His Glu Leu Ser Val Glu Gln Leu Thr Val 100105 110 tcg gag tac ctc cgc ttc aag gag gag ctt gtc gac ggc cag cac aat384 Ser Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Gln His Asn 115120 125 gat ccc tac gtt ctc gag ctt gac ttc gag ccg ttc aat gtc tca gtc432 Asp Pro Tyr Val Leu Glu Leu Asp Phe Glu Pro Phe Asn Val Ser Val 130135 140 cca cgc cca aat cgg tca tca tct att gga aac ggt gtg cag ttc ctc480 Pro Arg Pro Asn Arg Ser Ser Ser Ile Gly Asn Gly Val Gln Phe Leu 145150 155 160 aac cga cac ttg tcc tca atc atg ttc cgc aac agg gat tgc ttggag 528 Asn Arg His Leu Ser Ser Ile Met Phe Arg Asn Arg Asp Cys Leu Glu165 170 175 ccc ctg ttg gat ttc ctc cgt ggc cac cgg cac aag ggg cat gttatg 576 Pro Leu Leu Asp Phe Leu Arg Gly His Arg His Lys Gly His Val Met180 185 190 atg ctt aat gat aga ata caa agc ttg ggg agg ctt cag tct gtgctg 624 Met Leu Asn Asp Arg Ile Gln Ser Leu Gly Arg Leu Gln Ser Val Leu195 200 205 acc aaa gct gag gag cac ttg tca aag ctc cct gct gac aca ccatac 672 Thr Lys Ala Glu Glu His Leu Ser Lys Leu Pro Ala Asp Thr Pro Tyr210 215 220 tca caa ttt gct tat aaa ttt caa gag tgg ggc ctg gag aaa ggttgg 720 Ser Gln Phe Ala Tyr Lys Phe Gln Glu Trp Gly Leu Glu Lys Gly Trp225 230 235 240 ggt gat aca gca gga cat gtt ttg gaa atg atc cat ctc cttcta gac 768 Gly Asp Thr Ala Gly His Val Leu Glu Met Ile His Leu Leu LeuAsp 245 250 255 atc att cag gcg cca gac cca tct acc cta gag aaa ttc ttgggg agg 816 Ile Ile Gln Ala Pro Asp Pro Ser Thr Leu Glu Lys Phe Leu GlyArg 260 265 270 atc ccc atg att ttt aac gtt gtt gtg gta tcc cct cat ggatac ttt 864 Ile Pro Met Ile Phe Asn Val Val Val Val Ser Pro His Gly TyrPhe 275 280 285 ggt caa gct aat gta tta ggc ttg cca gac aca gga gga cagatc gtc 912 Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln IleVal 290 295 300 tat ata ctg gac caa gtc cgt gca cta gaa aat gag atg gttctc cgt 960 Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Asn Glu Met Val LeuArg 305 310 315 320 tta aag aaa caa ggg ctt gat gtt tcc cca aag att ctcatt gtt act 1008 Leu Lys Lys Gln Gly Leu Asp Val Ser Pro Lys Ile Leu IleVal Thr 325 330 335 cgg ctg ata cca gat gca aaa gga aca tca tgc aat cagcgg ctt gag 1056 Arg Leu Ile Pro Asp Ala Lys Gly Thr Ser Cys Asn Gln ArgLeu Glu 340 345 350 aga att agt gga aca cag cat act tac ata tta cga gttccc ttc aga 1104 Arg Ile Ser Gly Thr Gln His Thr Tyr Ile Leu Arg Val ProPhe Arg 355 360 365 aat gaa aat ggg ata ctt aag aaa tgg ata tca aga tttgat gtg tgg 1152 Asn Glu Asn Gly Ile Leu Lys Lys Trp Ile Ser Arg Phe AspVal Trp 370 375 380 cca tat ctg gaa aca ttt gct gag gat gct gct ggt gaaatt gct gct 1200 Pro Tyr Leu Glu Thr Phe Ala Glu Asp Ala Ala Gly Glu IleAla Ala 385 390 395 400 gaa tta caa ggt act cca gac ttc ata att gga aactac agt gat gga 1248 Glu Leu Gln Gly Thr Pro Asp Phe Ile Ile Gly Asn TyrSer Asp Gly 405 410 415 aat ctt gtg gcg tca ttg cta tct tac aag atg ggaatt acc cag tgc 1296 Asn Leu Val Ala Ser Leu Leu Ser Tyr Lys Met Gly IleThr Gln Cys 420 425 430 aac att gct cat gct ctg gaa aag act aag tat ccagat tca gac ata 1344 Asn Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro AspSer Asp Ile 435 440 445 ttt tgg aag aat ttc gat gag aag tac cat ttc tcctgc cag ttc act 1392 Phe Trp Lys Asn Phe Asp Glu Lys Tyr His Phe Ser CysGln Phe Thr 450 455 460 gct gat ata att gct atg aac aat gct gat ttt atcatc acc agc aca 1440 Ala Asp Ile Ile Ala Met Asn Asn Ala Asp Phe Ile IleThr Ser Thr 465 470 475 480 tac caa gaa att gct gga agc aaa aat act gttgga cag tat gag agt 1488 Tyr Gln Glu Ile Ala Gly Ser Lys Asn Thr Val GlyGln Tyr Glu Ser 485 490 495 cat act gcc ttt act ctg cct ggt ctg tac cgagtt gtc cat ggg atc 1536 His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg ValVal His Gly Ile 500 505 510 gat gtc ttc gat cca aag ttc aat ata gtc tctcct gga gct gac atg 1584 Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser ProGly Ala Asp Met 515 520 525 tcc ata tac ttt cca cat acc gag aag gcc aagcga ctc acc tct ctt 1632 Ser Ile Tyr Phe Pro His Thr Glu Lys Ala Lys ArgLeu Thr Ser Leu 530 535 540 cat ggt tca atc gaa aat ttg att tat gac ccggag caa aac gat gaa 1680 His Gly Ser Ile Glu Asn Leu Ile Tyr Asp Pro GluGln Asn Asp Glu 545 550 555 560 cac att ggg cat ctg gat gac cgg tca aagccc atc ctc ttc tcc atg 1728 His Ile Gly His Leu Asp Asp Arg Ser Lys ProIle Leu Phe Ser Met 565 570 575 gca aga ctc gac agg gtg aag aac ata acaggg ctg gtc gaa gct ttt 1776 Ala Arg Leu Asp Arg Val Lys Asn Ile Thr GlyLeu Val Glu Ala Phe 580 585 590 gct aag tgc gct aag ctg agg gag ctg gtaaac ctt gtc gtc gtt gcc 1824 Ala Lys Cys Ala Lys Leu Arg Glu Leu Val AsnLeu Val Val Val Ala 595 600 605 ggg tac aat gat gtc aac aag tcc aag gacagg gaa gag atc gcg gag 1872 Gly Tyr Asn Asp Val Asn Lys Ser Lys Asp ArgGlu Glu Ile Ala Glu 610 615 620 ata gag aag atg cat gaa ctc atc aag acccac aac ttg ttc ggg cag 1920 Ile Glu Lys Met His Glu Leu Ile Lys Thr HisAsn Leu Phe Gly Gln 625 630 635 640 ttc cgc tgg atc tct gcc cag aca aacagg gcc cgt aac ggc gag ctc 1968 Phe Arg Trp Ile Ser Ala Gln Thr Asn ArgAla Arg Asn Gly Glu Leu 645 650 655 tat cgc tac atc gct gat acc cat ggtgct ttc gta cag ccg gcc ttg 2016 Tyr Arg Tyr Ile Ala Asp Thr His Gly AlaPhe Val Gln Pro Ala Leu 660 665 670 tat gaa gcg ttc ggt ctc acc gtc gttgag gcc atg acc tgt ggg ctt 2064 Tyr Glu Ala Phe Gly Leu Thr Val Val GluAla Met Thr Cys Gly Leu 675 680 685 cct act ttc gcg acg ctc cat gga ggtcca gct gag atc ata gag cat 2112 Pro Thr Phe Ala Thr Leu His Gly Gly ProAla Glu Ile Ile Glu His 690 695 700 ggc gtc tcg ggc ttc cac att gac ccgtac cac ccc gaa cag gct gtt 2160 Gly Val Ser Gly Phe His Ile Asp Pro TyrHis Pro Glu Gln Ala Val 705 710 715 720 aat ctg atg gcc gac ttc ttc gaccgg tgc aag caa gac cca gat cac 2208 Asn Leu Met Ala Asp Phe Phe Asp ArgCys Lys Gln Asp Pro Asp His 725 730 735 tgg gtg aat ata tct gga gca gggctg cag cgc ata tac gag aag tac 2256 Trp Val Asn Ile Ser Gly Ala Gly LeuGln Arg Ile Tyr Glu Lys Tyr 740 745 750 aca tgg aag ata tac tca gag aggttg atg aca ctg gcc ggg gtc tac 2304 Thr Trp Lys Ile Tyr Ser Glu Arg LeuMet Thr Leu Ala Gly Val Tyr 755 760 765 ggt ttc tgg aag tac gtg tcg aagctc gag agg ctg gag acg agg cgc 2352 Gly Phe Trp Lys Tyr Val Ser Lys LeuGlu Arg Leu Glu Thr Arg Arg 770 775 780 tac ctt gag atg ttc tac ata ctgaag ttc cgc gag ctg gcg aag acc 2400 Tyr Leu Glu Met Phe Tyr Ile Leu LysPhe Arg Glu Leu Ala Lys Thr 785 790 795 800 gtg ccg ctt gca att gac caaccg cag tag cttgcgcaac tgcgactgcg 2450 Val Pro Leu Ala Ile Asp Gln ProGln * 805 tagcacttgg tacaagactg aaacctgaag gaccttcagt aatttaggcgcggcagacgg 2510 tagccaataa aatgtgccgg agctgaactg gttttttatt atgtacataatggcagtata 2570 acaaaattac tgaaggcagg tgggttgcag ttgtgtgttc gttactgtttactgtattat 2630 gtcaagctgt cggctgcaat ttctttgctg gcaagccgca ggcactggtgaagtgctgat 2690 aaatacatca tattctgttg acctgtgaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaggg 2750 cggccgc 2757 12 809 PRT Zea mays 12 Met Ser Ala Pro LysLeu Asn Arg Asn Ala Ser Ile Arg Asp Arg Val 1 5 10 15 Glu Asp Thr LeuHis Ala His Arg Asn Glu Leu Val Ala Leu Leu Ser 20 25 30 Lys Tyr Val AsnLys Gly Lys Gly Ile Leu Gln Pro His His Ile Leu 35 40 45 Asp Ala Leu AspGlu Val Gln Gly Ser Gly Val Arg Ala Leu Ala Glu 50 55 60 Gly Pro Phe LeuAsp Val Leu Arg Ser Ala Gln Glu Ala Ile Val Leu 65 70 75 80 Pro Pro PheVal Ala Ile Ala Val Arg Pro Arg Pro Gly Val Trp Glu 85 90 95 Tyr Val ArgVal Asn Val His Glu Leu Ser Val Glu Gln Leu Thr Val 100 105 110 Ser GluTyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Gln His Asn 115 120 125 AspPro Tyr Val Leu Glu Leu Asp Phe Glu Pro Phe Asn Val Ser Val 130 135 140Pro Arg Pro Asn Arg Ser Ser Ser Ile Gly Asn Gly Val Gln Phe Leu 145 150155 160 Asn Arg His Leu Ser Ser Ile Met Phe Arg Asn Arg Asp Cys Leu Glu165 170 175 Pro Leu Leu Asp Phe Leu Arg Gly His Arg His Lys Gly His ValMet 180 185 190 Met Leu Asn Asp Arg Ile Gln Ser Leu Gly Arg Leu Gln SerVal Leu 195 200 205 Thr Lys Ala Glu Glu His Leu Ser Lys Leu Pro Ala AspThr Pro Tyr 210 215 220 Ser Gln Phe Ala Tyr Lys Phe Gln Glu Trp Gly LeuGlu Lys Gly Trp 225 230 235 240 Gly Asp Thr Ala Gly His Val Leu Glu MetIle His Leu Leu Leu Asp 245 250 255 Ile Ile Gln Ala Pro Asp Pro Ser ThrLeu Glu Lys Phe Leu Gly Arg 260 265 270 Ile Pro Met Ile Phe Asn Val ValVal Val Ser Pro His Gly Tyr Phe 275 280 285 Gly Gln Ala Asn Val Leu GlyLeu Pro Asp Thr Gly Gly Gln Ile Val 290 295 300 Tyr Ile Leu Asp Gln ValArg Ala Leu Glu Asn Glu Met Val Leu Arg 305 310 315 320 Leu Lys Lys GlnGly Leu Asp Val Ser Pro Lys Ile Leu Ile Val Thr 325 330 335 Arg Leu IlePro Asp Ala Lys Gly Thr Ser Cys Asn Gln Arg Leu Glu 340 345 350 Arg IleSer Gly Thr Gln His Thr Tyr Ile Leu Arg Val Pro Phe Arg 355 360 365 AsnGlu Asn Gly Ile Leu Lys Lys Trp Ile Ser Arg Phe Asp Val Trp 370 375 380Pro Tyr Leu Glu Thr Phe Ala Glu Asp Ala Ala Gly Glu Ile Ala Ala 385 390395 400 Glu Leu Gln Gly Thr Pro Asp Phe Ile Ile Gly Asn Tyr Ser Asp Gly405 410 415 Asn Leu Val Ala Ser Leu Leu Ser Tyr Lys Met Gly Ile Thr GlnCys 420 425 430 Asn Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp SerAsp Ile 435 440 445 Phe Trp Lys Asn Phe Asp Glu Lys Tyr His Phe Ser CysGln Phe Thr 450 455 460 Ala Asp Ile Ile Ala Met Asn Asn Ala Asp Phe IleIle Thr Ser Thr 465 470 475 480 Tyr Gln Glu Ile Ala Gly Ser Lys Asn ThrVal Gly Gln Tyr Glu Ser 485 490 495 His Thr Ala Phe Thr Leu Pro Gly LeuTyr Arg Val Val His Gly Ile 500 505 510 Asp Val Phe Asp Pro Lys Phe AsnIle Val Ser Pro Gly Ala Asp Met 515 520 525 Ser Ile Tyr Phe Pro His ThrGlu Lys Ala Lys Arg Leu Thr Ser Leu 530 535 540 His Gly Ser Ile Glu AsnLeu Ile Tyr Asp Pro Glu Gln Asn Asp Glu 545 550 555 560 His Ile Gly HisLeu Asp Asp Arg Ser Lys Pro Ile Leu Phe Ser Met 565 570 575 Ala Arg LeuAsp Arg Val Lys Asn Ile Thr Gly Leu Val Glu Ala Phe 580 585 590 Ala LysCys Ala Lys Leu Arg Glu Leu Val Asn Leu Val Val Val Ala 595 600 605 GlyTyr Asn Asp Val Asn Lys Ser Lys Asp Arg Glu Glu Ile Ala Glu 610 615 620Ile Glu Lys Met His Glu Leu Ile Lys Thr His Asn Leu Phe Gly Gln 625 630635 640 Phe Arg Trp Ile Ser Ala Gln Thr Asn Arg Ala Arg Asn Gly Glu Leu645 650 655 Tyr Arg Tyr Ile Ala Asp Thr His Gly Ala Phe Val Gln Pro AlaLeu 660 665 670 Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr CysGly Leu 675 680 685 Pro Thr Phe Ala Thr Leu His Gly Gly Pro Ala Glu IleIle Glu His 690 695 700 Gly Val Ser Gly Phe His Ile Asp Pro Tyr His ProGlu Gln Ala Val 705 710 715 720 Asn Leu Met Ala Asp Phe Phe Asp Arg CysLys Gln Asp Pro Asp His 725 730 735 Trp Val Asn Ile Ser Gly Ala Gly LeuGln Arg Ile Tyr Glu Lys Tyr 740 745 750 Thr Trp Lys Ile Tyr Ser Glu ArgLeu Met Thr Leu Ala Gly Val Tyr 755 760 765 Gly Phe Trp Lys Tyr Val SerLys Leu Glu Arg Leu Glu Thr Arg Arg 770 775 780 Tyr Leu Glu Met Phe TyrIle Leu Lys Phe Arg Glu Leu Ala Lys Thr 785 790 795 800 Val Pro Leu AlaIle Asp Gln Pro Gln 805 13 347 DNA Sorghum propinquum 13 cgccagtcgccagtcgccac agccacacca caccacacta gccgcggccg cgggtaggag 60 cgcgcgcggcgcggcggaac gacccaccgg tggcggcagc catgtctgcc ccgaagctga 120 accgcaacgcgagcatccgg gaccgcgtcg aggacaccct ccacgcgcac cgcaacgagc 180 tcgtcgccctcctctccaag tacgtgaaca aggggaaggg catcctgcag ccgcaccaca 240 tcctcgacgcgctcgacgag gtccagggct ccggggtccg cgcgctcgcc gagggaccct 300 tcctcgacgtcctccgctcc gcgcaggagg cgatcgtgct gccgccg 347

What is claimed is:
 1. An isolated polynucleotide which encodes apolypeptide with sucrose synthase activity comprising a member selectedfrom the group consisting of: (a) a polynucleotide having at least 80%sequence identity, as determined by the GAP algorithm under defaultparameters, to a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 11; (b) apolynucleotide encoding a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 12;(c) a polynucleotide amplified from Zea mays nucleic acids using primerswhich selectively hybridize, under stringent hybridization conditions,to loci within a polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 11; (d) apolynucleotide which selectively hybridizes, under stringenthybridization conditions and a wash in 0.1×SSC at about 65° C., to apolynucleotide of SEQ ID NO: 1 or SEQ ID NO: 11; (e) a polynucleotide ofSEQ ID NO: 1 or SEQ ID NO: 11; (f) a polynucleotide which iscomplementary to a polynucleotide of (a), (b), (c), or (e); and (g) apolynucleotide comprising at least 50 contiguous nucleotides from apolynucleotide of (a), (b), (c), (d), (e), or (f).
 2. A recombinantexpression cassette, comprising a member of claim 1 operably linked, insense or anti-sense orientation, to a promoter.
 3. A host cellcomprising the recombinant expression cassette of claim
 2. 4. Atransgenic plant comprising a recombinant expression cassette of claim2.
 5. The transgenic plant of claim 4, wherein said plant is a monocot.6. The transgenic plant of claim 4, wherein said plant is a dicot. 7.The transgenic plant of claim 4, wherein said plant is selected from thegroup consisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, peanut, and cocoa.
 8. A seed fromthe transgenic plant of claim
 4. 9. A method of modulating the level ofsucrose synthase in a transgenic plant, comprising: (a) introducing intoa plant cell a recombinant expression cassette comprising apolynucleotide of claim 1 operably linked to a promoter; (b) culturingthe plant cell under plant cell growing conditions; (c) regeneratingsaid transgenic plant; and (d) expressing said polynucleotide, whichresults in production of an encoded protein, for a time sufficient tomodulate the level of sucrose synthase in said plant.
 10. The method ofclaim 9, wherein said plant is selected from the group consisting of:maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton,rice, barley, millet, peanut, and cocoa.
 11. The method of claim 9,wherein the encoded protein comprises a member selected from the groupconsisting of: (a) a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 12; (b) apolypeptide having at least 80% identity to, and having at least oneepitope in common with, a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 12,wherein said sequence identity is determined using the GAP algorithmunder default parameters; and (c) at least one polypeptide encoded by amember of claim
 1. 12. An isolated protein comprising a member selectedfrom the group consisting of: (a) a polypeptide of SEQ ID NO: 2 or SEQID NO: 12; (b) a polypeptide having at least 80% sequence identity to,and having at least one epitope in common with, a polypeptide of SEQ IDNO: 2 or SEQ ID NO: 12, wherein said sequence identity is determined bythe GAP algorithm under default parameters; and, (c) at least onepolypeptide encoded by a member of claim
 1. 13. A method of increasingcellulose production in the stalk tissue of a transgenic plant,comprising: (a) introducing into a plant cell a recombinant expressioncassette comprising a sucrose synthase polynucleotide operably linked toa promoter; (b) culturing the plant cell under plant cell growingconditions; (c) regnerating said transgenic plant; and (d) expressingsaid polynucleotide for a time sufficient to increase the level ofsucrose synthase in said plant.
 14. The method of claim 13, wherein saidplant is selected from the group consisting of: maize, soybean,sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley,millet, peanut, and cocoa.
 15. The method of claim 13, wherein saidsucrose synthase polynucleotide is Sus1, Sh1, or Sus3 from maize. 16.The method of claim 13, wherein said promoter preferentially directsexpression in stalk tissue.
 17. A method of increasing the concentrationof cellulose in the tissues of a seed of a transgenic plant, comprising:(a) introducing into a plant cell a recombinant expression cassettecomprising a sucrose synthase polynucleotide operably linked to apromoter; (b) culturing the plant cell under plant cell growingconditions; (c) regnerating said transgenic plant; and (d) expressingsaid polynucleotide for a time sufficient to increase the level ofsucrose synthase in said seed of said transgenic plant.
 18. The methodof claim 17, wherein said plant is selected from the group consistingof: maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton,rice, barley, millet, peanut, and cocoa.
 19. The method of claim 17,wherein said sucrose synthase polynucleotide is Sus1, Sh1, or Sus3 frommaize.
 20. The method of claim 17, wherein said promoter preferentiallydirects expression in the seed.
 21. The method of claim 17, wherein saidpromoter preferentially directs expression in the pericarp.